WO2006104639A2 - Device comprising array of micro-or nano-reservoirs - Google Patents

Abstract

A device comprised of one or more micro- or nano-reservoirs is described. Each reservoir is accessible via a nanoscale aperture, or nanopore. The reservoirs may be loaded with one or more reactants or agents, for release in response to a stimulus or used in other microfluidic applications.

Description

DEVICE COMPRISPJG ARRAY OF MICRO- OR NANO-RESERVOIRS

Cross Reference To Related Applications

This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/666,668 filed on March 29, 2005, which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to a device and a method to deliver small quantities of biologically active species with extremely high spatial and temporal accuracy. In particular, the invention relates to a device comprising an array of micro- or nano-scale reservoirs etched into a substrate. The reservoirs can be filled with different drugs or biological signaling agents which are released on demand to the surrounding environment.

Background of the Invention

Research on cell behavior on artificial materials has shown the local microenvironment around a cell, including bound, soluble and mechanical signals, regulate a variety of cellular phenomena including differentiation, cell motility, and proliferation. Multiple signals are generally present at one time, and their effect can differ depending upon their concentration and method of presentation. For instance, fibroblast adhesion to a surface coated with an extra-cellular matrix (ECM) integrin adhesion ligand, RGD, increases upon spatial clustering of the ligands relative to a homogenous distribution (Maheshwari, G., et al. (2000) J. Cell Science 113:1677-1686; Maheshwari, G., et al. (1999) Biophysical J. 76:2814-2823). Addition of a soluble signal, epidermal growth factor (EGF), can either increase or decrease the locomotion speed depending on the concentration of RGD presented on the surface (Maheshwari, et al. (1999) supra). CHO cell migration has been shown to depend upon three separate variables: surface-bound ligand level, integrin expression level, and integrin-ligand binding affinity (Palecek, S. P., et al. (1997) Nature 385:537-540). Similarly, myoblast adhesion and differentiation on RGD-treated surfaces depends on the concentration of both RGD and Ca²⁺ (Rowley, J. A, et al. (2002) Adv.

Materials 14:886-889). These studies used homogenous presentations of the soluble factors, yet it is known that keratocyte mobility uses spatially localized Ca²⁺ transients to help coordinate the necessarily asymmetric cellular processes responsible for locomotion (Doyle, A., et al. (2004) J. Cell Science, 117:2203-2214). Clearly, control over the composition and spatial presentation of signaling factors is a necessary consideration for cell behavior and differentiation.

Biomaterials researchers have investigated a number of methods to present the correct ECM or growth factors, with varying levels of spatial and temporal control. The general strategy is to take a known bioactive molecule and place it in the desired location by: (a) grafting/patterning it on a flat surface (Spargo, B. J., et al. (1994) Proceedings of the National Academy of Sciences of the USA, 91:11070-11074; Chen, C. S., et al. (1997) Science 276:1425-1428; Chen, C. S , et al. (1998) Bio. Progress 14:356-363; Kane, R. S., et al. (1999) Biomaterials 20:2363-2376; Mrksich, M., et al. (1996) Proceedings of the National Academy of Sciences of the USA, 93:10775-10778); (b) attaching it to a 3D polymer matrix or hydrogel (Maheshwari, et al. (2000) supra; Kapur, T. A. and Shoichet, M. S. (2004) J. Biomedical Materials Research, 68A:235-243; Zisch, A. H., et al. (2001) J. Controlled Release 72:101-113); (c) incorporating it in a degradable polymer (Uhrich, K. E., et al. (1999) Chemical Reviews 99:3181-3198; Lee, K. Y. and Mooney, D. J. (2001) Chem. Rev. 101:1869-1879; Griffith, L. G. (2000) Acta Materialia 48:263-77; Patel, N., et al. (1998) FASEB Journal 12:1447-1454); or (d) embedding it within a polymer with known diffusion characteristics. (Uhrich, et al. (1999) supra; Lee, et al. (2001); Sakiyama-Elbert, S. E. and Hubbell, J. A. (2000) J. of Controlled Release, 69:149-158). For instance, it has been demonstrated that for cells cultured on a surface patterned with small islands of fibronectin, cell apoptosis rates decreases as the size of the island increased-thus the spatial presentation of the integrin receptors can dictate cell behavior (Chen, et al. (1997) supra; Chen, et al. (1998) supra). Cells have been patterned by various techniques by treating a selected area with adhesive ligands and surrounding it with protein resistant molecules (e.g. poly ethylene glycol) (Folch, A. and Toner, M. in ANNUAL REVIEW OF BIOMEDICAL ENGINEERING; V.2; eds. Yarmush, M. L., Diller, K. R. & Toner, M., pp. 227-256 (2000)). However, these systems present static signals or predetermined release rates, which may not be appropriate for influencing the developmental journey of multipotent cells. For epi- genetic engineering of complicated structures like tissues, it is desirable to have sub-sets of cells directed down separate paths simultaneously (Rowley, et al. (2002) supra; Alsbsrg, E., et al. (2002) Proceedings of the National Academy of Sciences of the USA, 99:12025- 12030).

One method to achieve this is to externally control the factor presentation and release characteristics with electrical signals. This has been demonstrated using micro- reservoirs of drugs that are released by electrochemically dissolving a gold film covering the reservoir (Santini, J. T., et al. (1998) Nature, 397:335-8). In this manner, -25 nL aliquots of drug stored within 50 μm x 50 μm pores could be released on command. Solutions of Ca²⁺, sodium fluorescein, and poly(ethylene glycol) were successfully released, requiring roughly an hour to fully distribute into solution. However, the large size of the reservoirs precluded accurate spatial control of local drug concentration.

In parallel with new biomaterials-based approaches, researchers have improvised a number of ways to interface microelectronics with single cells. These techniques include chip-based patch clamps (Farre, C, et al. (2001) Biophysical J. 80:338a-338A); Olofsson, J., et al. (2003) Biophysical J. 84:488a-488A; Schmidt, C, et al. (2000) Angewandte Chemie-International Edition, 39:3137-3140), nanoscale Si nozzles, (Lehnert, T., et al. (2002) Applied Physics Letters 81:5063-5065) vesicle coated transistors (Rentschler, M. and Fromherz, P. (1998) Langmuir 14:547-551; Fromherz, P., et al. (1999) Applied Physics A (Materials Science Processing) A69:571-6), and neural interfaces (Andersson, H. and Van Den Berg, A. (2004) Current Opinion in Biotechnology 15:44-49; Fromherz, P. (2002) Chemphyschem 3:276-284). Single ion channel activity (Schmidt, et al. (2000) supra, neuron signaling (Fromherz (2002) supra), or cell viability were measured using these methods (Huang, Y., et al. (2003) Sensors and Actuators A (Physical) A105:31-39).

However, there remains a need for a device that can store a biologically active agent and that will release the agent or agents on command. In particular, there remains a need for a device that store one or more biologically active agents in a cavity or reservoir having dimensions less than an average biological cell, so that a cell when in contact with the device is in fluid communication with a plurality of the reservoirs. Release of the same or different agents from the reservoirs, on command, provides a unique and powerful approach to localized bioactive-agent delivery that will advance drug delivery, development of biomaterials that rely on cell differentiation, and epigenetic cell-based therapies. Summary of the Invention

In one aspect, the invention includes a device having one or more micro- or nano- reservoir(s), each reservoir accessible by an aperture, such as a nanopore.

In one embodiment, a gate or fluid regulating means is disposed over the nanopore.

In other embodiments, the device is comprised of a base composed of a first material and of a second material adjacent the base, the reservoir being formed in one of the materials and the nanopore formed in the other material.

In another embodiment, electrodes are in contact with one or both of the materials to provide an electric field to or through the gate.

The invention also includes a method for delivering one or more agents to a localized area by providing a device as described herein.

In a further aspect, the invention includes a population of isolated cells cultivated on or over a substrate comprising micro- and/or nano-scale reservoirs.

In yet a further aspect, the substrate contains one or more reservoirs having an average volume of less than about 10 pL (10^"n L) and a gate or fluid regulating means for reversibly accessing the reservoirs.

In another aspect, the method can be used to prepare a pre-determined tissue by contacting an isolated stem or progenitor cell with a substrate or device as described above under conditions suitable for the expansion of the stem or progenitor cell into the predetermined tissue composition.

In a further aspect, the invention is an in vitro method to assay for modulation of the expansion of an isolated stem cell or an isolated population of stem cells by contacting the isolated stem cell or the isolated population of stem cells on the substrate as described above and in the presence of a test agent and monitoring the effect of the test agent on the expansion of the isolated stem cell or the isolated population of stem cells.

These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings. Brief Description of the Drawings

Figs. IA- IB are schematic illustrations of exemplary devices with nanoscale or microscale reservoirs in fluid communication with a nanopore that opens to the external environment;

Figs. 2A-2F are illustrations of a single reservoir in a device (Fig. 2A) and with various optional gates overlying the pore for control over the release of the reservoir contents;

Figs. 3 A-3B are illustrations of an exemplary reservoir with a lipid bilayer membrane gate (Fig. 3A) and an array of such reservoirs on a chip, each reservoir being electronically addressable;

Fig. 3C shows the device of Fig. 3 A with an applied voltage to the lipid bilayer gate;

Figs. 4A-4B illustrate approaches to fabrication of exemplary devices;

Fig. 5 is a computer- generated scanning electron micrograph image of pores ranging in size from 100 nni to 1 μm, formed by focused ion beam etching;

Figs. 6A-6B are computer-generated scanning electron micrograph images taken at various angles of a single reservoir;

Figs. 7A-7D are computer-generated scanning electron micrograph images of a reservoir having a hemispherical shape, the reservoir shown in top view (Fig. 7A) and in cross-sectional view at different magnifications (Fig. 7B, 7C, 7D);

Figs. 8A-8C are computer-generated scanning electron micrograph images of arrays of reservoirs of various sizes;

Fig. 9 A illustrates preparation of a reservoir structure comprised of a reservoir and a nanopore and a lipid membrane gate;

Figs. 9B and 9C are optical fluorescence images, 100Ox magnification, of a device showing a reservoir-nanopore structures with and without a BLM gate fabricated from fluorescently-labeled lipids;

Figs. 10A- 1OD are fluorescence images from a fluorescence recovery after photobleaching analysis of a reservoir/nanopore structure having a bilayer lipid membrane gate;

Fig. 11 is an optical micrograph of a device taken 14 hours after fluorescently- labeled lipid vesicles were trapped in the reservoirs and sealed with a bilayer lipid membrane gate; Figs. 12A-12F are fluorescent optical images of two reservoir structures, the reservoir structure on the lower left containing a fluorescent dye, the images taken at two minute intervals;

Figs. 13A-13D are illustrations of a process for loading a reservoir with an agent and depositing a bilayer lipid membrane gate over the nanopore;

Fig. 14 is an two-color fluorescence image of an array of reservoir structures having fiuorescently-labeled vesicles trapped in most of the reservoirs, the device prepared according to the process of Figs. 13A-13D; and

Fig. 15 is an illustration of a device separated into four quadrants for release of different agents into each quadrant to influence differentiation of cells growing in each quadrant.

Figs. 16A-16E and 17A-17C are side cross sectional views of steps in a method of forming and removing an electrochemical seal.

Figs. 18A, 18B and 18C are three dimensional and side cross sectional views, respectively, of devices according to embodiments of the invention.

Figs. 19A-19C are SEM micrographs of an array of nanopores. Figure 19A is an optical image of an array of 1 micron diameter pores etched through a Pt electrode (white background) and into a Si wafer. Figure 19B is an SEM image of gold reduced from solution onto the electrode to seal the pores. Variations in the pulse voltage, length, and wait between voltage pulses can prevent the reduced gold from 'flowering' out of the orifice. This deposition occurred at -0.5 V (pulsed for 100 ms and recovered for 900 ms for 100 cycles). Figure 19C is an SEM image of the same pores after seal dissolution in a Ix PBS solution. A potential of +1.1 V was applied for 180s to fully dissolve the Au.

Detailed Description of the Embodiments of the Invention

As used herein, certain terms have the following defined meanings.

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

A "reservoir" as used herein shall mean a volume where a fluid is retained or held, generally implying that this space is distinguishable from the surrounding media or materials.

A micro-reservoir intends a reservoir where the diameter of the reservoir is in the 1 μm to 50 μm range, or the fluid volume is approximately between 0.1 fL to 10 pL.

A nano-reservoir intends a reservoir where the diameter of the reservoir is in the 10 nm to 1000 nm range, or the fluid volume is approximately between 0.1 zL to 0.1 fL..

As used herein, the term "nanopore" shall mean a hole or channel connecting a reservoir to another fluid volume, with dimensions between 10 nm and 10 μm.

"Substrate" is the material upon which the device is fabricated, and may or may not comprise the reservoirs or fluid gates.

A "gate or regulating means" disposed over the substrate shall mean any means capable of regulating fluid flow, either convective, driven (eg electrokinetic) or diffusive, between two volumes connected by a channel.

The term "isolated" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, are normally associated with in nature. For example, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated with in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. For example, an isolated cell refers to a cell that has been removed from its native host and unless specifically recited, the term "isolated cell" is intended to refer to a single cell or a population of cells. As used herein, "stem cell" defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells. At this time and for convenience, stem cells are categorized as somatic (adult) or embryonic. A somatic stem cell is an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated. An embryonic stem cell is a primitive (undifferentiated) cell from the embryo that has the potential to become a wide variety of specialized cell types. An embryonic stem cell is one that has been cultured under in vitro conditions that allow proliferation without differentiation for months to years. A clone is a line of cells that is genetically identical to the originating cell; in this case, a stem cell.

"Clonal proliferation" refers to the growth of a population of cells by the continuous division of single cells into two identical daughter cells.

As used herein, a "pluripotent cell" defines a less differentiated cell that can give rise to at least two distinct (genotypically and/or phenotypically) further differentiated progeny cells.

A "multi-lineage stem cell" or "multipotent stem cell" refers to a stem cell that reproduces itself and at least two further differentiated progeny cells from distinct developmental lineages. The lineages can be from the same germ layer (i.e. mesoderm, ectoderm or endoderm), or from different germ layers. An example of two progeny cells with distinct developmental lineages from differentiation of a multilineage stem cell is a myogenic cell and an adipogenic cell (both are of mesodermal origin, yet give rise to different tissues). Another example is a neurogenic cell (of ectodermal origin) and adipogenic cell (of mesodermal origin).

"Differentiation" describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. "Directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type. "Dedifferentiated" defines a cell that reverts to a less committed position within the lineage of a cell. As used herein, the term "differentiates or differentiated" defines a cell that takes on a more committed ("differentiated") position within the lineage of a cell. As used herein, "a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage" defines a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively. Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal.

Examples of cells that differentiate into ectodermal lineage include, but are not limited to epidermal cells, neurogenic cells, and neurogliagenic cells.

Examples of cells that differentiate into endodermal lineage include, but are not limited to pleurogenic cells, and hepatogenic cells, cell that give rise to the lining of the intestine, and cells that give rise to pancreogenic and splanchogenic cells.

As used here, "adipose tissue" defines a diffuse organ of primary metabolic importance made up of white fat, yellow fat or brown fat. The adipose tissue has adipocytes and stroma. Adipose tissue is found throughout the body of an animal. For example, in mammals, adipose tissue is present in the omentum, bone marrow, subcutaneous space and surrounding most organs.

"Adipose-Derived Stem Cell" (ADSC) is an adult stem cell that is or has a parental cell that was obtained from a tissue source containing adipose tissue.

As used herein and as set forth in more detail below, "conditioned medium" is medium which was cultured with a mature cell that provides cellular factors to the medium such as cytokines, growth factors, hormones, and extracellular matrix. A medium that has been exposed to mature myoctytes is used to culture and induce ADSCs to differentiate into a myogenic lineage. Culruring in a medium conditioned by exposure to heart valve cells can induce differentiation into heart valve tissue. ADSCs cultured in a medium conditioned by neurons can be differentiated into a cell of the neuronal lineage. Cells culture in medium conditioned by hepatocytes can induce differentiation into cells of the endodermal lineage.

As used herein, an "antibody" includes whole antibodies and any antigen binding fragment or a single chain thereof. Thus the term "antibody" includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, any of which can be incorporated into an antibody of the present invention. The term "antibody" is further intended to encompass digestion fragments, specified portions, derivatives and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and CH, domains; a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V_H and CH, domains; a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, a dAb fragment (Ward et al. (1989) Nature 341:544-546), which consists of a V_H domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et.al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Single chain antibodies are also intended to be encompassed within the term "fragment of an antibody." Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

A "composition" is also intended to encompass a combination of active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di~, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffmose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

The term carrier further includes a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, fϊcolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2- hydroxypropyl-.quadrature.-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives and any of the above noted carriers with the additional provisio that they be acceptable for use in vivo. For examples of carriers, stabilizers and adjuvants, see Martin REMINGTON'S PHARM. SCL, 15th Ed. (Mack Publ. Co., Easton (1975) and Williams & Williams, (1995), and in the "PHYSICIAN'S DESK REFERENCE", 52^nd ed., Medical Economics, Montvale, NJ. (1998).

An "effective amount" is an amount sufficient to effect beneficial or desired results whether it is therapeutic or diagnostic. An effective amount can be administered in one or more administrations, applications or dosages.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. In one aspect, this invention provides a population of isolated cells that are cultivated or cultured on or over a semiconductor substrate comprising micro- and/or nano- scale reservoirs. It is intended that any viable cell can be grown, cultured or cultivated on this substrate. The cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human. Suitable mammalian cells include, but are not limited to stem cells, clonal stem cells, a substantially homogenous population of progenitor cells, a substantially homogenous population of expanded differentiated cells and a heterogenous population of progenitor cells or a heterogenous population of expanded differentiated cells. When the isolated, single cell is a stem cell, the substrate is particularly suited for the growing, culturing or propagating of one or more embryonic stem cells or somatic stem cells. The substrate is particularly suited for the propagation of one or more stem cells because the cells will reproduce in the absence of a feeder layer that is typically required. Accordingly, in one aspect, the invention provides a population of isolated cells that are cultivated or cultured on or over a semiconductor substrate comprising micro- and/or nano-scale reservoirs and in the absence of a feeder layer.

Presently, somatic stem cells are described by the tissue from which they are isolated and in some cases, further by certain cell surface markers. Stem cells have been isolated from hemaotpoeitic tissue (hematopoietic stem cells or "HSCs"), mesenchymal tissue (mesenchymal stem cells or "MSC") and adipose tissue (Adipose-derived stem cells or "ADSCs"), for example. See Verfaillie, C. "Adult Stem Cells: Tissue Specific or Not ?", Handbook of Stem Cells, Vol. 2 (2004), supra. Any of these stem cells types can be grown or cultivated on the substrate of this invention.

As is known to those of skill in the art, stem cells are unique in that they possess the ability to reproduce themselves and the ability to differentiate into cells of a pre-determined lineage depending on the microenvironment of the cell. Thus, when the microenvironment, e.g., the solution within the micro- or nano-reservoir contains neurogenic differentiation medium, the cell in contact with that medium will differentiate into a cell within that lineage, a neuronal progenitor cell or for example, a fully differentiated nerve cell. Due to the nano-scale of the reservoirs within the substrate, one or more cells adjacent to that reservoir can contain the same or different cell culture medium. Thus, one can create a differentiated cell population having a complex composition that is present in tissue in vivo.

For a general description of stem cells see HANDBOOK OF STEM CELLS, Vol. I and II (2004), Elsevire Inc. and the National Institute of Health web page available at the address "stemcellsnih.gov".

Thus, in one aspect, the substrate can contain the same medium in each reservoir and the cells grown or cultivated on the substrate will evolve into a substantially homogenous population of cells. As used herein, the term "substantially homogenous" is intended to mean greater than 70 %, or alternatively greater than 75 %, or alternatively greater than 80 %, or alternatively greater than 85 %, or alternatively greater than 90 %, or alternatively greater than 95 %, or alternatively greater than 97% of the identical cell type. In some aspect, the reservoirs contain medium that promotes the stem cells to expand into a clonal population of stem cells, i.e., cell that retain the ability to reproduce and further differentiate. Because of the clonal nature of the cells, an expanded population of clonal cells are substantially homogenous in that each "stem cell" is an identical copy of its parent stem cell.

The devices and methods of this invention can also be utilized to proliferate, expand and/or differentiate embryonic stem cells. Embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro and then donated for research purposes. There is still dispute in the scientific community over the identifying characteristics of embryonic stem cells but the following are some of the characteristics published by the National Institutes of Health (NIH) on their web site which can be accessed at the web address "stemcells.nih.gov/info/basics/basics3.asp" (last accessed on January 25, 2006):

1) the presence of a protein called Oct-4, which undifferentiated cells typically make. Oct-4 is a transcription factor;

2) the phenotype, when examined microscopically should show that the chromosomes are unchanged and undamaged; and

3) evidence of pluripotentency by 1) allowing the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so they will differentiate to form specific cell types; or 3) injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types — an indication that the embryonic stem cells are capable of differentiating into multiple cell types. Methods to isolate embryonic stem cells have been reported in the patent and technical literature. For example, U.S. Patent No. 6,280,718, issued August 28, 2001 discloses methods of obtaining human pluripotent embryonic stem cells using mammalian stromal cells. See also U.S. Patent No. 6,200,806, issued March 13, 2001. U.S. Patent Appl. No.: 20050260747, published November 24, 2005 discloses methods to obtain and culture undifferentiated human embryonic stem cells as well as methods and compositions to differentiate the cells in vitro. U.S. Patent Appl. No.: 20060015961, published January 19, 2006, discloses the isolation and use of peripheral blood derived germ stem cells. Embryonic stem cell lines are also available through WiCeIl Research Institute, Madison Wisconsin, which can be accessed through the web site at the address "wicell.org" (last accessed on January 25, 2006).

Materials for manufacturing the substrates include, but are not limited to semiconductor substrates, such as silicon, germanium or compound semiconductor wafers, with an optional passivation layer, such as polymers (eg PMMA or epoxy), photoresists (eg Shipley resists or Microchem SU-8), molecular coatings (Silgard), or oxide layers (eg SiO₂ or Al₂O₃). The passivation layer can be comprised of any material that will electrically isolate electrodes from any external solution, e.g. SU-2 or SU-8 photoresist. Alternatively, the substrate does not have to contain a semiconductor material. For example, the substrate may comprise a polymeric, glass, ceramic, quartz or metallic material. Preferably, the substrate comprises two or more layers, one of which has distinct etching or removal characteristics than the others (i.e., where one layer may be selectively or preferentially etched compared to the other layer). However, the substrate may comprise a single "layer", such as a semiconductor, ceramic, metal, glass, quartz or polymeric wafer or plate containing the reservoirs. Such single layer substrate may be attached to other layers after the formation of the reservoirs. For example, the single layer substrate may be used for microfluidic devices described in more detail below. Such single layer microfluidic substrate may be bonded to polymeric layer or layers after the formation of the reservoir(s).

Within the substrate, one or more micro- or nano-reservoirs of approximate volume of about 1 aL to about 1 pL or about 5 pL or alternatively about 100 pL.

In another aspect, the substrate contains a gate or fluid regulating means for reversibly accessing the reservoirs. The gates or fluid regulating means allows reversible access to a pore having a width of less than about 10 μm. Suitable gates or regulating means include, but are not limited to a lipid membrane, a flap, a valve, a bubble or an electrochemically deposited seal.

In a further aspect, the substrate contains (a) a base composed of a first material and of a second material adjacent to the base and (b) the reservoir being formed in one of the materials and the pore formed in the other material. In another aspect, the electrode is in contact with one or both of the materials to provide an electric field to or through the gate. In another aspect, the gate or regulating means comprises an active electrode located in or adjacent to the opening and a counter electrode and the active electrode is adapted to form at least a partial seal over the reservoir when the substrate is placed into a solution or a suspension and a voltage is applied between the active electrode and the counter electrode to electrochemically deposit a material from the solution or suspension in the reservoir.

In a further aspect, the active electrode is adapted to electrochemically remove the seal when the substrate is placed into a solution or a suspension and a opposite polarity voltage is applied between the active electrode and the counter electrode. The gate or regulating means can be open or closed over the reservoir.

The reservoir or reservoirs can contain a liquid or fluid composition, e.g., a pharmaceutical composition or growth or cell culture medium. When the fluid is a cell culture medium, it may be desirable to include one or more of an agent selected from the group consisting of a growth medium, a biologically active agent.

The substrate can be used to differentiate stem cells or clonally propagate them. Thus, this invention also provides methods for cultivating or propagating stem cells by contacting one or more isolated cells or populations of cells with a substrate as described above or a device as described infra, under conditions that are suitable for the growth or propagation of the cells. Suitable compositions for this use in connection with the differentiation of stem cells are described infra. Examples of such include, but are not limited to adipogenic differentiation medium, osteogenic differentiation medium, leiomyogenic differentiation medium, chondrogenic differentiation medium, myogenic differentiation medium or neuronal differentiation medium.

This substrate can be used to clonally grow or differentiate adispose-derived stem cells. Methods to separate, isolate and expand ADSCs are known in the art and described, for example in U.S. Patent Nos. 6,391,2971Bl; 6,777,231Bl; Bums et al. (1999) MoI Endocrinol 13:410-7; Erickson et al. (2002) Biochem Biophys Res Commun. Jan. 18, 2002;

290(2):763-9; Gronthos et al. (2001) Journal of Cellular Physiology, 189:54-63; Halvorsen et al. (2001) Metabolism 50:407-413; Halvorsen et al. (2001) Tissue Eng. 7(6):729-41; Harp et al. (2001) Biochem Biophys Res Commun 281 :907-912; Saladin et al. (1999) Cell Growth & Diff 10:43-48; Sen et al. (2001) Journal of Cellular Biochemistry 81:312-319; Zhou et al. (1999) Biotechnol. Techniques 13: 513-517; Erickson et al. (2002) Biochem Biophys Res Commun. Jan. 18, 2002; 290(2):763-9; Gronthos et al. (2001) Journal of Cellular Physiology, 189:54-63; Halvorsen et al. (2001) Metabolism 50:407-413; Halvorsen et al. (2001) Tissue Eng. Dec. 7, 2001; (6):729-41; Harp et al. (2001) Biochem Biophys Res Commun 281:907-912; Saladin et al. (1999) Cell Growth & Diff 10:43-48; Sen et al. (2001) Journal of Cellular Biochemistry 81:312-319; Zhou et al. (1999) Biotechnol. Techniques 13:513-517; Zuk et al. (2001) Tissue Eng. 7: 211-228.

ADSCs can be obtained from any animal (alive or dead) so long as adipose stromal cells within the animal are viable. Suitable tissue sources of ADSCs include, but are not limited to any fat-containing tissue, e.g., brown or white adipose tissue such as subcutaneous white adipose tissue. Typically, human adipose tissue is obtained from a living donor using surgical excision or suction lipectomy. Alternatively, a mechanical system such as described in U.S. Pat. No. 5,786,207 to Katz et al is used. Paragraph [0200] of U.S. Patent App. Publ. No. 2005/0076396Al, details another appropriate protocol for the separation and isolation of ADSCs. Briefly, the authors of the patent publication stated that a hollow blunt-tipped cannulae was introduced into the subcutaneous space through small (N 1 cm) incisions of a patient undergoing elective liposurgery. The cannulae was attached to a gentle suction and moved through the adipose compartment, mechanically disrupting the fat tissue. A solution of saline and the vasoconstrictor, epinephrine, was infused into the adipose compartment to minimize blood loss and contamination of the tissue by peripheral blood cells. The raw lipoaspirate (approximately 300 cc) was processed according to established 10 methodologies in order to obtain the stromal vascular fraction (SVF). Hauner et al. (1987) J. Clin. Endocrinol. Metabol. 64: 832-835; Katz et al. (1999) Clin. Plast. Surg. 26: 587-603, viii. The authors reported that to isolate the SVF, lipoaspirates were washed extensively with equal volumes of Phospho-Buffered Saline (PBS) and the extracellular matrix (ECM) was digested at 37° C for 30 minutes with 0.075% collagenase. Enzyme activity was neutralized with Dulbecco's Modified Eagle's Medium (DMEM), containing 10% Fetal Bovine Serum (FBS) and centrifuged at 1200 X g for 10 minutes to obtain a high-density SVF pellet. The pellet was resuspended in 160 mM NH₄CI and incubated at room temperature for 10 minutes to lyse contaminating red blood cells. As reported by the authors, the SVF was collected by centrifogation, as detailed above, filtered through a 100 Nm nylon mesh to remove cellular debris and incubated overnight at 37 ° Cl 5% CO₂ in Control Medium (DMEM, 10% FBS, 1% antibiotidantimycotic solution). Following incubation, the plates were washed extensively with PBS to remove residual nonadherent red blood cells to obtain a slurry of cells. The authors also reported that the cells were maintained at subconfluent levels to prevent spontaneous differentiation.

ADSCs in the slurry can be isolated to multiple single cells or a substantially homogeneous composition by methods that include, but are not limited to, cell sorting, size fractionation, granularity, density, molecularly, morphologically, and immunohistologically using methods described in the art and infra for ADSCs, as well as methods described for other stem cells with minor modifications apparent to those of skill in the art. Such methods are described for hematopoietic stem cells (Yoon et al. (2005) J. Clin. Invest. 115(2):326-338; Camargo et al. (2003) Nat. Med. 9(12):1520-1527; and Matsuzaki et al. (2004) Immunity 20:87-93), mesenchymal stem cells (WO 97/18299), and pancreatic stem cells (Suzuki et al. (2004) Diabetes 53:2143-2152).

For the purpose of illustration only, several morphological, biochemical or molecular-based methods are used to isolate the cells. In one aspect, ADSCs are isolated based on cell size and granularity since ADSCs are small and agranular. Alternatively, because stem cells tend to have longer telomeres than differentiated cells, ADSCs can be isolated by assaying the length of the telomere or by assaying for telomerase activity.

Alternatively, ADSCs can be separated from the other cells of the pellet immunohistochemically by selecting for ADSC-specific cell markers using methods suitable materials and methods, e.g., panning, using magnetic beads, or affinity chromatography.

In one embodiment, the stem cells are cultured without differentiation using standard cell culture media, referred to herein as control medium, (e.g., DMEM, typically supplemented with 5-15% serum (e.g., fetal bovine serum, horse serum, etc.). The stem cells can be passaged at least five times or even more than twenty times in this or similar medium without differentiating to obtain a substantially homogeneous population of ADSCs. The ADSCs can be identified by phenotypic identification. To phenotypically separate the ADSCs, the cells are plated at any suitable density which may be anywhere from between about 100 cells/cm² to about 100,000 cells/cm² (such as about 500 cells/cm² to about 50,000 cells/cm², or, more particularly, between about 1,000 cells/cm² to about 20,000 cells/cm²).

For the purpose of illustration only, a preferred culture condition for cloning stem cells comprises about 213 F₁₂ medium+20% serum (preferably fetal bovine serum) and about 113 standard medium that has been conditioned with stromal cells or 15% FBS, 1% antibiotic/antimycotic in F-12/DMEM [1 :1]) (e.g., cells from the stromal vascular fraction of liposuction aspirate, the relative proportions can be determined volumetrically).

In addition to the experimental example discussed infra, paragraphs [0179] and [0180] of U.S. Patent Appl. Publ. No. 2005/0076396Al describes the cloning of ADSCs. Briefly, the authors reported that PLA cells were plated at about 5,000 cells/100 mm dish and cultured and that after some rounds of cell division, some clones were picked with a cloning ring and transferred to wells in a 48 well plate. As reported, the medium was changed twice weekly and the cells were cultured for several weeks until they were about 80% to about 90% confluent (at 37° C. in about 5% CO₂ in 213 F₁₂ medium +20% fetal bovine serum and 113 standard medium that was first conditioned by cultured PLA obtained following the protocol described in Example 1 and referred to as the "cloning medium"). Thereafter, the authors reported that each culture was transferred to a 35 mm dish and grown, and then retransferred to a 100 mm dish and grown until close to confluent. Following this, the authors report that one cell population was frozen, and the remaining populations were plated on 12 well plates, at 1000 cells/well.

In Paragraph [0180], the authors reported that cells were cultured for more than 15 passages in cloning medium and monitored for differentiation as indicated in Example 1 in the published application. The undifferentiated state of each clone remained true after successive rounds of culturing.

ADSCs, whether clonal or not, can be cultured in a specific inducing medium to induce the ADSC to differentiate and express its multipotency. The ADSCs give rise to cells of smooth muscle, mesodermal, ectodermal and endodermal lineage, and combinations thereof. Thus, one or more ADSCs derived from a multipotent ADSC can be treated to differentiate into a variety cell types.

To induce leimyogenic differentiation, ADSCs can be cultured in inductive medium (Medium MCDB 131 (Sigma, MI)) supplemented with 1% FBS plus 100 U/ml heparin). Alternatively the media can be supplemented with 2% FBS and 200U/ml heparin. When differentiated in vitro, the cells can be cultured in the presence of an effective amount of laminin, collagen, and/or heparin.

To induce adipogenic differentiation, ADSCs can be cultured in media containing a glucocorticoid (e.g., dexamethasone, hydrocortisone, cortisone, etc.), insulin, a compound which elevates intracellular levels of cAMP (e.g., dibutyryl-CAMP, 8-CPT-CAMP (8- (4)chlorophenylthio)-adenosine 3', 5' cyclic monophosphate; 8-bromo-CAMP; dioctanoyi- CAMP, forskolin etc.), and/or a compound which inhibits degradation of CAMP (e g , a phosphodiesterase inhibitor such as isobutyl methyl xanthine (IBMX), methyl isobutylxanthine, theophylline, caffeine, indomethacin, and the like), and serum. Thus, exposure of the ADSCs to between about 1 μM and about 10 μM insulin in combination with about 10 ^"9 μM to about 10^"6 M to (e.g., about 1 μM) dexamethasone can induce adipogenic differentiation. Such a medium also can include other agents, such as indomethacin (e.g., about 100 μM to about 200 μM), if desired, and preferably the medium is serum-free. Alternatively, ADSCs cultured in DMEM, 10% FIBS, 1 μM dexamthasone, 10 μM insulin, 200 μM indomethacin, 1 % antibiotic / antimicotic, (ABAM), 0.5 mM IBMX, take on an adipogenic phenotype.

ADSCs cultured in a composition comprising about 10^"7 M and about 10^"9 M dexamethasone (e.g., about 1 Ah) in combination with about 10 μM to about 50 μM ascorbate-2-phosphate and between about 10 nM and about 50 nM /^-glycerophosphate are induced to differentiate into cells of the osteogenic lineage. The medium can further include serum (e.g., bovine serum, horse serum, etc.). An alternative medium contains DMEM, 10% FBS, 5% horse serum, 50 μM hydrocortisone, 10^"7 M dexamethosone, 50 μM ascorbate-2-phosphate, 1% ABAM.

ADSCs cultured in a composition comprising between about 10 μM and about 100 μM hydrocortisone, preferably in a serum-rich medium (e.g., containing between about 10% and about 20% serum (either bovine, horse, or a mixture thereof)) will differentiate into cells of the myogenic lineage. Other glucocorticoids that can be used include, but are not limited to, dexamethasone. Alternatively, 5'-azacytidine can be used instead of a glucocorticoid. Alternatively, ADSCs cultured in DMEM, 10% FIBS, 10 ^"7M dexamethosone, 50 nM ascorbate-2-phosphate, 10 mM beta-glycerophosphate, 1% ABAM, take on an myogenic phenotype.

To induce chondrogenic differentiation, cells can be cultured in a composition comprising between about 1 μM to about 10 μM insulin and between about 1 μM to about 10 μM transferrin, between about 1 ng/ml and 10 ng/ml transforming growth factor (TGF) β 1, and between about 10 nM and about 50 nM ascorbate-2-phosphate (50 nM). For chondrogenic differentiation, preferably the cells can be cultured in high density (e.g., at about several million cells/ml or using micromass culture techniques), and also in the presence of low amounts of serum (e.g., from about 1 % to about 5%).

ADSCs cultured in DMEM, 50 ~Mascorbate-2-phosphate, 6.25 pg/ml transferin, 10 ng/ml insulin growth factor (IGF-I), 5 ng/ml TGFβ-1, 5 ng/ml basic fibroblast growth factor (bFGF; used only for one week), are induced to differentiate into cells of the chondrogenic phenotype.

To induce ectodermal (e.g., neurogenic) differentiation, ADSCs can be cultured in a medium comprising DMEM, no serum and 5-10 mM . jS-mercaptoethanol and assume an ectodermal lineage.

Differentiated cells can be induced to dedifferentiate into a developmentally more immature phenotype (e.g., a fetal or embryonic phenotype) by co-culturing the cells with cells isolated from fetuses or embryos, or in the presence of fetal serum.

Methods to characterize differentiated cells that develop from the ADSCs, include, but are not limited to, histological, morphological, biochemical and immunohistochemical methods, or using cell surface markers, or genetically or molecularly, or by identifying factors secreted by the differentiated cell, and by the inductive qualities of the differentiated ADSCs. Molecular characterization of differentiated ADSCs can be achieved by measurement of telomere length since undifferentiated stem cells have longer telomeres than differentiated cells. Alternatively or in addition, the cells can be assayed for the level of telomerase activity.

Molecular markers can be used to identify cell types. For the purpose of illustration only, mesodermal lineage markers include, but are not limited to, MyoD, myosin, α-actin, brachyury, xFOG, Xtbx5 FoxFl, XNkx-2.5. Ectodermal lineage molecular markers include but are not limited to N-CAM, GABA and epidermis specific keratin. Molecular markers that characterize cells of the endodermal lineage include, but are not limited to, Xhbox8, Endol, Xhex, Xcad2, Edd, EFl- a, HNF3-j8, LFABP, albumin, insulin. Unless otherwise stated, mammalian homologs of the markers can be used.

Detection of molecular markers can be by any appropriate method, including for example, detecting the quantity of mRNA transcribed from the marker gene or the quantity of cDNA produced from the reverse transcription of the mRNA transcribed from the gene or the quantity of the polypeptide or protein encoded by the gene. These methods can be performed on a sample by sample basis or modified for high throughput analysis. Additionally, databases containing quantitative full or partial transcripts or protein sequences isolated from a cell sample can be searched and analyzed for the presence and amount of transcript or expressed gene product.

In assaying for an alteration in mRNA level, nucleic acid contained in a sample of cells is first extracted according to standard methods in the art. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures known in the art or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures. The mRNA of a gene contained in the extracted nucleic acid sample is then detected by hybridization (e.g., Northern blot analysis) and/or amplification procedures according to methods widely known in the art.

In certain embodiments, it will be advantageous to employ nucleic acid probe or primer sequences in combination with an appropriate means, such as a label, for detecting hybridization and therefore complementary sequences. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. One can employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

This invention further provides a device having one or more micro- or nano- reservoir(s), each reservoir being accessible by an aperture, such as a nanopore. In one aspect, the device also has a gate or a fluid regulating means disposed over the nanopore. The gate or the regulating means comprises at least one of a reversible membrane, such as a lipid membrane, a flap, a valve, a bubble or an electrochemically deposited seal, o a stimulus.

In a further aspect, the device is comprised of a base composed of a first material and of a second material adjacent to the base and the reservoir(s) is formed in one of the materials and the nanopore formed in the other material. In one embodiment, the electrodes are in contact with one or both of the materials to provide an electric field to or through the gate.

In an alternate embodiment, the invention provides a device having an opening located between two communicating volumes; an active electrode located in or adjacent to the opening; and a counter electrode; wherein the active electrode is adapted to form at least a partial seal in the opening when the device is placed into a solution or a suspension and a voltage is applied between the active electrode and the counter electrode to electrochemically deposit a material from the solution or suspension in the opening. In one embodiment, the active electrode is adapted to electrochemically remove the seal when the device is placed into a solution or a suspension and a opposite polarity voltage is applied between the active electrode and the counter electrode.

This invention also provides a device, comprising an opening located between two communicating volumes; a counter electrode; a first means for forming at least a partial seal in the opening when the device is placed into a solution or a suspension and a voltage is applied between the first means and the counter electrode to electrochemically deposit a material from the solution or the suspension in the opening. In one aspect, the first means also is a means for electrochemically removing the seal when the device is placed into a solution or a suspension and a voltage is applied between the first means and the counter electrode.

The invention further provides a device having an opening located between two communicating volumes; an active electrode located in or adjacent to the opening; a counter electrode; and a first means for applying a voltage between the active electrode and the counter electrode to electrochemically deposit a material from a solution or suspension in the opening to at least partially seal the opening. In one aspect, the first means is also a means for applying an opposite polarity voltage between the active electrode and the counter electrode to electrochemically remove the seal when the device is placed into a solution or a suspension.

In another aspect, the invention provides a device having an opening located between two communicating volumes; a seal located in the opening; an active electrode located in or adjacent to the opening; and a counter electrode, wherein the active electrode is adapted to electrochemically remove the seal when the device is placed into a solution or a suspension and a voltage is applied between the active electrode and the counter electrode. The invention also provides a device having an opening located between two communicating volumes; a seal located in the opening; a counter electrode; and a first means for electrochemically removing the seal when the device is placed into a solution or a suspension and a voltage is applied between the first means and the counter electrode.

In another aspect, the invention provides a device having comprising an opening located between two communicating volumes; a seal located in the opening; an active electrode located in or adjacent to the opening; a counter electrode; and a first means for applying a voltage between the active electrode and the counter electrode to electrochemically remove the seal when the device is placed into a solution or a suspension.

This invention also provides an electrochemical sealing method, the method requiring placing a device into a solution or a suspension, wherein the device comprises an opening located between two communicating volumes, an active electrode located in or adjacent to the opening, and a counter electrode; and applying a voltage between the active electrode and the counter electrode to deposit a material from the solution or the suspension in the opening to at least partially seal the opening, hi one aspect, the opening is unsealed (opened) by applying a voltage of an opposite polarity between the active electrode and the counter electrode to remove the deposited material from the opening. Alternatively, the invention provides a electrochemical unsealing method by placing a device into a solution or suspension, wherein the device comprises at least a partial seal located in an opening which is located between two communicating volumes, an active electrode located in or adjacent to the opening, and a counter electrode; and applying a voltage between the active electrode and the counter electrode to remove the seal from the opening.

This invention further provides an in vitro method to assay for modulation of the expansion of an isolated stem cell or an isolated population of stem cells by contacting the isolated stem cell or the isolated population of stem cells on the semiconductor substrate as described above and in the presence of a test agent and monitoring the effect of the test agent on the expansion of the isolated stem cell or the isolated population of stem cells.

I. Device

In one aspect, the invention provides a device comprising one or more reservoir structure, each reservoir structure comprised of a cavity or reservoir in communication with a pore that provides access to an external environment. As used herein, the term "reservoir structure" intends a reservoir in combination with a pore, as illustrated in, for example, Figs. 2A-2B, discussed below. Exemplary embodiments of the device comprising a plurality of reservoir structures are illustrated in Figs. 1 A-IB. The illustrations show a device comprised of a base layer, typically a silicon wafer, having a plurality of reservoirs etched into the base. A second layer is disposed over the base, and an individual aperture or opening that is typically of nanoscale dimensions, hence is referred to herein as a nanopore, is provided in the second layer for access to each reservoir. Multilayer stacks are also possible; the requirement is for two of the layers to have different etching properties. As seen in Fig. IA and in Fig. 2 A, the nanopore can be flush with the surface of the device, or as seen in Fig. IB and in Fig. 2B, can be recessed from the surface of the device. The spacing between the nanopores is defined by the patterning technique used, and as shown in Fig. IA, in one embodiment, can be as small as 100 nm.

The device can optionally include a cell adhesion area, as indicated in Fig. IB, to facilitate seeding and growth of cells on the surface of the device. The device can optionally include electrodes for electrical stimulation of each reservoir, pore, or optional gate which is described in more detail with reference to Figs. 2C-2F.

Each reservoir can contain one or more agents, which is used here in its broadest sense to be any agent that may have no effect, a beneficial effect, or detrimental effect on a biological cell. Exemplary agents are given below. In principle, any compound can be stored and delivered upon command with nanometer spatial resolution as will be further described below.

With reference now to Figs. 2A-2F, various embodiments of an optional gate disposed over the nanopore are shown in Figs. 2C-2F. As mentioned above, Fig. 2 A shows a reservoir accessible by a nanopore that does not include the optional gate. Fig. 2B also shows a reservoir accessible by a nanopore that does not include the optional gate, the nanopore being recessed from the surface of the device. It will be appreciated that either embodiment can include the optional gate. Fig. 2C shows a nanopore with a reversibly permeable membrane disposed over the pore, to control access of fluid to and from the reservoir. One example of a reversibly permeable membrane is a lipid bilayer membrane, and in studies conducted in support of the invention devices having an array of reservoirs were prepared, each reservoir accessible via a nanopore that included a bilayer lipid membrane gate. These devices will be described below. Other suitable gates can take the form of a simple mechanical flap (Fig. 2D), a mechanical valve (Fig. 2E), or a bubble (Fig. 2F). Mechanical flaps and valves are well known in the microfhiidics technology field. Those of skill can readily envision variations on the gates illustrated in Figs. 2C-2F.

The gate in general is at or near the pore opening and serves to control flow into and out of the reservoir. Since the pore itself is quite small, the sealing operation is fast and efficient. The valve can also be recessed to avoid direct contact with any cells or species on the upper surface. The combination of the gate and the reservoir concept allows controlled storage and release of a number of agents in the reservoir and permits spatially and temporally controlled release of the agents.

Fig. 3 A illustrates an exemplary reservoir with a lipid bilayer membrane gate. A plurality of such reservoirs can be patterned onto a suitable base material to form an array of reservoirs in a device, such as the one shown in Fig. 3B. Each reservoir in the array in accessible by a nanopore, which in this embodiment has dimensions of 100 nm-10 μm. The depth of the reservoir in this embodiment is between 0.5-10 μm. A bilayer lipid membrane is disposed over the nanopore, to act as a fluid regulating means to control the flow of material to and from the reservoir. Electrical contact is made to the base layer, here a silicon layer, and a reference electrode in solution. If desired, physical or chemical barriers can be patterned to farm lipid corrals to hold specific membrane proteins within the reservoir area. The reservoir is filled with an agent, such as a drug solution.

Fig. 3 C illustrates electroporation of the bilayer lipid membrane gate to release the contents of the reservoir. A voltage of ~100-500 mV is applied to the base silicon layer, causing charged layers to build up across the membrane. The voltage opens aqueous pores within the membrane, allowing charged ions and drug to flow out of the reservoir. Typically these pores are 1-5 nm in diameter, and remain open for milliseconds to seconds depending upon the amplitude and length of the applied voltage pulse.

The device described above can be prepared by the exemplary processes shown in Fig. 4A and in Fig. 4B. In general, and with reference to Fig. 4 A, a silicon (Si) base with roughly 25-100 nm of oxide is used as the starting wafer, the Si being a first material and the SiO₂ being a second material. The silicon can be patterned into electrodes if desired by initially using a silicon-on-insulator wafer and patterning with photolithography, or by patterned doping of the silicon. A nanoscale pore is etched through the SiO₂ layer using a Focused Ion Beam (FIB). Alternatively, a pore geometry can be patterned with photolithography or e-beam lithography, followed by reactive ion etching to create the pore.

A number of alternative means of patterning and forming the nanopore could be envisioned by someone familiar with the art. Then, a reservoir is selectively etched into the Si layer with a selective etchant, such as KOH or XeF₂ etch. The substrate is rilled with drug and coated with a gate, such as a bilayer lipid membrane (BLM) by vesicle rupture from aqueous solution containing the desired concentration of reagent. Alternatively, the drug may be loaded after BLM deposition via electroporation. Then the surface of the device is washed to provide a device ready for operation.

The nanopore is made through the SiO₂ layer and is in fluid communication with the reservoir within a Si layer. It will be appreciated that the reservoir can be larger than the nanopore or can also be of nanoscale dimensions. The nanopore and reservoir are made by nanoscale etching though the SiO₂ followed by a selective Si etch. The base substrate can be, for example, a standard p- or n- doped <100> silicon wafer (Silicon Quest International, Santa Clara, CA) with a sheet resistance less than 1 Ω/cm². Alternatively, a Silicon-On- Insulator (SOI) or SIMOX substrate wafer can be used (Silicon Quest International). In this process, the silicon layer on top of the silicon oxide can be lithographically patterned to form individual contacts to each device. After patterning the silicon, all the subsequent processing steps are identical for both substrates. To form the SiO₂ layer on top of the silicon, the upper 20-100 nm of Si is thermally oxidized in a 900° C oxidation furnace, as shown in step (a) of Figure 4A. A deposited rather than a thermally grown oxide layer may be used instead. Furthermore, materials other than silicon oxide, such as silicon nitride or aluminum oxide, which can be act as a mask during the selective or preferential etching of the underlying substrate may be used instead. Alternatively, as shown in steps (a) and (b) of Figure 4B, a trench may be etched into the silicon substrate prior to the oxidation. For example, the trench may be about 0.1 to about 0.5 microns deep and about 0.5 to about 2 microns wide.

As shown in step (b) of Figure 4A, etching of ~50-1000 nm nanopores though the SiO₂ is achieved by, for example, using a Focused Ion Beam (FEI Strata 235 Dual Beam SEM/FIB) to ablate the oxide with high energy Ga ions. This process is fast (~ 10 s for a 50 nm oxide), one-step, easily patterned, and can produce holes as small as 50 nm. Fig. 5 shows a scanning electron micrograph image of pores ranging in size from 100 nm to 1 μm, formed by focused ion beam etching. The holes formed have a slight bevel around the edges, and may be rougher than those etched with reactive ions. Reactive ion etched (RIE) holes can be made by defining circles in ZEP-520 resist (Zeon Corporation, Tokyo, Japan) with e-beam lithography, followed by etching with a 5:1 SF₆:O₂ plasma etch or other etching media, such as CF₄. This approach will give sharper side walls around the pore, but the surface of the silicon may become modified by the resist and etching process.

The silicon reservoirs are formed by selectively etching the Si with either KOH, TMAH, or XeF₂. The etch rate for Si is ~1500 A/min for these etchants, much larger than that for SiO₂ (< 1 A/min). The rapid etching of the underlying Si layer results in an undercut SiO₂ membrane, as shown in step (c) of Figure 4A. If the trench shown in Figure 4B is present, then the membrane is recessed from the upper surface of the substrate, as shown in step (d) of Figure 4B. KOH is anisotropic, etching the Si (100) plane faster than the (110) plane, producing pyramidal etch pits, as shown in the scanning electron micrograph images in Figs. 6A-6C. Etching in 18M KOH may be conducted for about 1 minute at 8O⁰C. XeF₂ is a high-vapor pressure solid (~ 3.8 torr at 25° C) that isotropically etches silicon in the gas phase due to attack by F₂ to gaseous form SiF₄. XeF₂ is advantageous because the reaction takes place entirely in the gas phase, eliminating stiction problems associated with wet chemical processing. The XeF₂ etch can be conducted for about 30 seconds. The reservoirs may be about 1 to about 5 microns deep and about 1 micron in diameter, for example.

The shape and volume of the reservoir can be varied by the etching method selected and the conditions of the etch. Figs. 6A-6C show images of reservoirs resulting from anisotropic etching of the Si base with KOH. The reservoirs can be etched using an isotropic etch to yield hemispherical cavities, as shown in the scanning electron micrograph images of Figs. 7A-7D. Isotropic etching can be accomplished by, for example, tetramethyl hydroxide (TMAH) etching or xenon difluoride (XeF₂) etching. Fig. 7A is a top view image of a reservoir, the nanopore opening visible as the dark spot. Cross-sectional views of the reservoir and the nanopore, at different magnifications are shown in Fig. 7B, 7C, and 7D.

The density of the array of nanopores and reservoirs, which together form a structure, is controlled by the lithographic technique, as can be appreciated by those skilled in these techniques. The size of the reservoir is controlled with the etch conditions, including concentration of etchants, time of etching, and temperature. Figs. 8A-8C are computer-generated scanning electron micrograph images of arrays of reservoirs of various sizes. The reservoirs in Fig. 8A are rather large, on the order of 3 mm. The reservoirs in Figs. 8B and 8C on 2 μm and 5 μm, respectively. The dark spot in the center of each reservoir is the nanopore, typically having dimensions of about 300 nm. Various devices were constructed to illustrate the fabrication and use. Devices were prepared according to the procedure illustrated in Figs. 4A-4B. If desired, an optional cell adhesion layer or matrix is formed on the substrate. For example, as shown in step (e) of Figure 4B, the cell adhesion layer may be formed by coating the cell adhesion layer material onto a stamp, such as a PDMS stamp, followed by placing the substrate in contact with the stamp. If the trench is present in the substrate, then the stamp may be shaped to place the cell adhesion layer on the surface of the substrate adjacent to the trench, but not in the trench.

Then, a bilayer lipid membrane (BLM) gate was deposited onto the nanopore of each reservoir/nanopore structure, as shown in steps (d) and (e) of Figure 4A. The BLM gate was deposited by reconstituting dried lipids in water, homogenizing them into uniform size unilamellar vesicles, and rupturing them on the silicon chip, as illustrated in Fig. 9A. Low transition temperature lipids were used to avoid crystallization during handling, specifically egg-derived phosphatidylcholine (eggPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) (Avanti Polar Lipids, Alabaster, AL). A small amount (~1 mol%) of Texas Red dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) or l-oleoyl-2-[6-[(7-nitro-2- l,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphoethanolamine (NBD-PE) fluorescent lipid dye (Molecular Probes, Eugene, Oregon) was incorporated into the lipid membrane to allow fluorescence imaging of the BLM after deposition. More specifically, lipid vesicles for deposition were prepared by hydrating the phospholipids with a rehydration buffer solution (10 mM Tris, 100 mM NaCl, pH 8, or drug-containing solution) to give large, multilamellar vesicles. Suspensions of the multilamellar lipid vesicles were homogenized into smaller unilamellar vesicles either by bath sonication or repeated extrusion through a small hole (e.g., a 100 nm extruder from Avanti Polar Lipids). A carefully cleaned silicon substrate (15 min hot piranha, Dl H₂O rinse, baked 2 hrs at 400° C, cooled to room temperature) was placed on top of a small drop of vesicle solution. Due to electrostatic interactions between the surface and the lipids, the vesicles ruptured and fused onto the silicon oxide surface. Figs. 9B and 9C are optical fluorescence images, 100Ox magnification, of a device showing a reservoir-nanopore structures with and without a BLM gate. As shown in Figures 4A and 4B, the reservoirs are filled and emptied from an opening in the same (i.e., top) side of the substrate. The reservoirs are also etched from that same (i.e., top) side of the substrate. Alternatively, the reservoirs may be etched and filled from the opposite (i.e., bottom) side of the substrate from the side from which they are emptied. In this case, a sealing layer is formed on the bottom side of the substrate to seal the reservoirs after they are filled. The gates are still formed on the top side of the substrate and are used to controllably release the material from the reservoirs.

II. Exemplary Device Utilities

The substrates and devices described herein are useful to culture and expand, in a controlled manner a population of isolated cells such as stem cells by contacting the cells with a substrate or device having loaded in the reservoirs an effective amount of a preselected composition. Compositions and other bioactive reagents can be loaded into the nanoreservoirs in at least two ways, now to be described. If the same medium or drug is to be loaded into all the reservoirs, as for initial testing, the desired reagent is supplemented into the re-hydration solution for the lipids before deposition on the chip. When the chip is placed in contact with the unilamellar solution, the medium will naturally fill all the reservoirs due to capillary action. Fusion of the vesicles onto the surface will seal the solution inside. Release of reagents from the reservoir can be assessed using the technique described in Example 2. In brief, a calcium sensitive fluorophore Fluo-5N (Molecular Probes) is loaded into each reservoir at 5 μm concentration for subsequent imaging of the reservoir and drug release. Alternatively, different drugs and media can be stored into each nanoreservoir by electroporating the correct solution through the lipid layer after the membrane has been deposited, hi this approach, a universal buffer solution is initially stored in all the reservoirs, and the reservoirs sealed by the lipid membrane. The desired medium for a sub-set of reservoirs is then pipetted on top of the chip, and those reservoirs electroporated to introduce the medium into the pores. After 1 minute the pores in the lipid membrane re-seal and the chip is be rinsed thoroughly to remove the medium. The process is then repeated until all the reservoirs contain the correct solution. Removal of drugs from the exterior of the membrane after each step is tested by filling the reservoirs with Fluo-5N, rinsing, then introducing a 10 mM Ca²⁺ solution on top of the chip. Any residual Fluo-5N will fluoresce brightly when exposed to calcium levels above 50 μM, thus indicating incomplete flushing of the supernatant.

Adhesion of the lipid membrane to the silicon can be modified through changing the electrostatics or hydrophilicity of the silicon oxide surface before lipid deposition. Reducing the negative surface charge density on the oxide layer often increases lipid adhesion. For example, the surface of the device can be functionalized with either poly-L- lysine or 4-aminobutyldimethylmethoxy silane, (ABDMS) to increase the lipid seal resistances up to 74 GΩ and >200 GΩ , respectively. Other surface modifications include thin coatings of cellulose, dextran, or alkanes.

The stability of the lipid membrane on the nanopore chip can be quantified by the length of time until the lipid membrane either (a) ruptures or (b) loses fluidity. The continuity and fluidity of the lipid membrane were evaluated based on the diffusivity of the lipids in the membrane as measured from fluorescence recovery after photobleaching (FRAP). The results are shown in Figs. 10A- 10D. In this technique, the aperture of the microscope is reduced to illuminate only a small spot of the lipid membrane (-50 μm), and the lipid fluorophores in this area irreversibly were bleached by exposure to high-intensity photoexcitation (diode laser or HBO mercury lamp, Carl Zeiss). The dark spot in the image of Fig. 1OA corresponds to the bleached lipid fluorophores. The photoexcitation was then reduced to a level below which it did not damage the lipids, the aperture opened, and the fluorescence intensity of the spot measured periodically (Axiocam HRm camera with Axio Vision 4.0 time-lapse module, Carl Zeiss). Figs. lOB-lOC shows the fluorescent images at progressively longer times after irreversibly bleaching of the lipid fluorophores. The images show that the membrane lipids are mobile (i.e., the membrane is fluid) since the bleached lipids gradually diffused out of the bleached area, while un-bleached lipids diffused in. The net effect was an increase in the fluorescence intensity of the spot as a function of time, with faster lipid diffusion giving faster recovery kinetics. The two- dimensional diffusion constant, D, of the lipids based on the mathematical analysis of the fluorescence recovery was calculated to be between 1 x 10^"8 and 1 x 10^"9 cm²/sec.

Devices having an array of reservoir structures were prepared as described above. Fluorescent lipid vesicles of about 700 nm in diameter were prepared and deposited on the surface of the device. Optical micrographs of the device surface were taken, and an image is shown in Fig. 11. This image was taken 14 hours after the lipid vesicles were trapped in the reservoirs and sealed with a bilayer lipid membrane gate. Figs. 12A-12F are fluorescent optical images of two reservoir structures, the reservoir structure on the lower left containing a fluorescent dye. The images are taken at two minute intervals and illustrate the diffusion of the dye from the reservoir, evidenced by the decreasing fluorescence intensity with increasing time.

Figs. 13 A-13D illustrate a process for loading a reservoir structure with a desired agent and fabricating a lipid bilayer membrane gate over the nanopore. A suspension of lipid vesicles, liposomes or micelles or other lipid structure, is deposited on the surface of a device containing at least one reservoir structure, as illustrated in Fig. 13 A. The lipid vesicles rupture and form a bilayer lipid membrane over the surface of the device. The lipid vesicles are of a size less than the diameter of the nanopore, so that the bilayer lipid membrane does not form across the nanopore. A solution or suspension containing an agent is then deposited on the device, as shown in Fig. 13B. Next, and with reference to Fig. 13C, a solution of lipid vesicles is deposited, where the vesicles have a size equal to or greater than the size of the nanopore. The vesicles rupture when contact with the edge of a nanopore is made, forming a bilayer lipid membrane gate over the nanopore, as illustrated in Fig. 13D.

A two-color fluorescence image of an array of reservoir structures prepared according to the process of Figs. 13A-13D is shown in Fig. 14. Most of the reservoirs of each reservoir structure contain a fluorescent dye with a lipid bilayer membrane gate disposed over the nanopore. The reservoirs that did not hold dye were mechanically ruptured from previous processing; thus 100% yield of dye loading can be achieved.

Localized release of the reservoir contents can be accomplished by electroporating the lipid membrane gate disposed on or over the nanopore. Electroporation parameters can be optimized to deliver 5 μM Fluo-5N dye solution from the reservoirs into a high calcium concentration solution (10 mM) and quantified with fluorescence microscopy (Zeiss Axioskop 2FS, 25x magnification, NA=0.75, water immersion objective). For example, a 1-10 ms square wave voltage pulse 100-500 mV in amplitude may be applied from a Keithley 6430 sub-femtoamp sourcemeter with an external preamp located next to the sample. Electrical contact between the sourcemeter and the sample is made using micromanipulators with blunt-tip electrical probes (Probing Solutions, Dayton, NV) on a custom-built probe-station/fluorescence microscope apparatus. The experiment is controlled with a computer equipped with a GPIB card to drive and collect data from the

6430 sourcemeter. Immediately after the electroporation pulse, the current due to ion flow across the membrane is measured with sub-femtoamp sensitivity. Expected current due to ion motion in 0.1 M NaCl solution due to ion migrating through the porous lipid membrane is -1-10 pA.

The magnitude, length, and number of repetitions of square wave pulses can be systematically varied to optimize dye release, as quantified from fluorescence microscopy. Previous studies of single cell electroporation using patch-clamp apparatus found pulse widths of 1-2 ms with an amplitude of 5.6 V and 100 Hz frequency resulted in efficient electroporation of fluorescein dye and DNA fragments into a-TN4 cells using a 440 nm diameter pipette tip (Rae, J. L et al., Pfluegers Archiv European Journal of Physiology, 443:664-670 (2002)). Using these as baseline values, the electrical parameters can readily be varied to optimize release of Fluo-5N into a calcium rich environment. An electrophysiology microscope (Axioskop 2FS, Carl Zeiss) equipped with a sensitive camera (Axiocam HRm, Carl Zeiss) can be used to acquire fluorescence image

By way of specific example, the delivery of soluble growth factors from the nanopore device can be used to influence the differentiation pathway of ADSCs. To illustrate that differentiation arises from the signals delivered from the nano-reservoirs, the chip can contain four different regions, each with different differentiation supplements within the reservoirs. Note there will be no physical barrier or differences between the regions- they are distinguished only by what growth factors are stored within the reservoirs. Thus if cells within each region differentiate down separate pathways, the differentiation can be clearly attributed to the signals released from the nanoreservoirs. Fig. 11 shows a drawing of a device with four such separate regions. Each of the reservoirs in the four regions can be loaded with different differentiation media to induce for example, osteogenic, adipogenic or non-specific differentiation of stem cells can be cultured on top of the chip in a non-differentiation inducing buffer solution or in medium that promotes expansion of clonal stem cells.

By way of example only, ADSCs are initially cultured to confluence 4-5 days in Dulbecco's modified Eagle's medium (DMEM). The cells are harvested by digestion with 0.5 mM EDTA/0.05% trypsin, centrifuged at 1,200 rpm for 5 min, re-suspended in DMEM and quantified. These cells are then seeded immediately on a substrate or device. The growth factor supplements for each quadrant are selected based on their anticipated ability to cause differentiation. Examples of differentiation media are as follows:

The adipogenic and osteogenic media have been shown to differentiation in vitro. For example, Wnt5a can induce osteogenic differentiation in the absence of BMP and retinoic acid. Other possible media include chondrogenic and myogenic solutions. The differentiation media will be sequentially loaded into the appropriate nano-reservoirs by electroporation in the presence of the correct differentiation solution. Between each loading the chip is thoroughly washed three times with control solution to remove any residual medium from the previous loading. After filling the nano-reservoirs, 50 μL of AMC cells are seeded into the cell-culture area of a sterilized nanoreservoir chip. The cells are allowed to adhere for 2 h, then 100 μL of control growth medium added.

Cell differentiation within the culture well is evaluated after 3, 7, and 14 days as a function of the amount of supplement delivered. In a first study, one-hundred 1 μm x 1 μm x 1 μm nano-reservoirs per electrode is used, for a total volume of 0.1 pi per electrode. To increase the amount of medium delivered, multiple electrodes can be electroporated at one time. Electroporation can be performed at the same time for all regions. The media is delivered every 8 hrs in small quantities such that an average concentration in each region can be established. For 3, 7, and 14 day cultures (common periods for differentiating AMCs), 36, 84, and 168 electrodes will be required, which can be easily fabricated using photolithography. Contacts to the electrodes are made with a printed circuit probe card (Probing Solutions, Dayton, NV), and multiplexed with a Keithley 7002 switching mainframe with five 40-channel 7001 matrix cards, for a total of 200 independent channels. A computer with a GPIB card controls delivery of the reagents automatically. After the proscribed time of 3, 7, or 14 day, the amount of cell differentiation as a function of location cells is evaluated within the culture well. The cells in the well are washed three times with 150 mM NaCl, fixed in ice-cold 70% ethanol, rinsed with distilled water, and stained with 30 μL of 2% Alizarin Red and 30 μL of 2% Oil Red O dye. The cells are incubated with the dyes for 10 minutes, and then rinsed with 70% ethanol to remove excess dye and three rinses of distilled water. The amount of differentiation will be evaluated from fluorescence microscopy using Alizarin Red and Oil Red O dyes, which have been shown to be selective indicators for osteogenic and adipogenic differentiation in AMCs.

These device described herein has a wide variety of applications in biological sciences. The reservoir structures (reservoir plus nanopore) can be patterned into various spatial configurations for localized drug delivery for the study of biological processes in the presence of heterogeneous media delivered from the reservoirs via the nanopores. For example, various cell-specific differentiation media could be delivered at different parts of a device to stimulate multi-cell line tissues to develop, such as a bone-cartilage construct that could be used to treat cartilage disorders. Concentration gradients are known to be important in biological systems, yet are difficult to create controllably on-chip. This device would enable large concentration gradients to be established by controlled fluid release. Alternatively, bio-derived sensors, such as lipid-bound protein receptors could be suspended over the pores, and the electrical current measured as protein-binding reactions occur at the surface. For small pore sizes, individual protein binding events can be recorded. The device also finds use in providing nanoscale reaction chambers or sampling containers.

Commercialization possibilities include supplying researchers in biology with a 'test bed' on which to tightly control the local chemical conditions, creating a sensor array chip, or a multi-well chip for combinatorial chemical reactions.

From the foregoing, it can be seen how various objects and features of the invention are met. A device and a method for delivering small quantities of biologically active species with extremely high spatial and temporal accuracy is provided. The device finds particular use in delivering biochemical cues to cells to effect, for example, cell differentiation, tissue regeneration, nerve signaling, etc. by providing a controlled platform where the biochemical signals are controllable in both a spatial and temporal manner. An array of nanoscale or microscale reservoirs etched into a based material, such as silicon, are filled with the same or different drugs or biological signaling agents. The reservoirs can be sealed with a gate to regulate the flow of the agent from the pore. Activation of the gate releases the agent on command. Each gate can be activated independently, if desired. The device offers significant advantages over functionalized polymer scaffolds loaded with drugs, as the chemical release is entirely controlled, with spatial resolution smaller then a cell diameter (-200 nm spacing), and temporal resolution on the order of seconds, and preferably less than one second.

In summary, the device described provides a means to selectively deliver bioactive reagents with unprecedented spatial and temporal control. The device is capable of providing the necessary agents to guide the differentiation of multipotent cells with environmental signals. The nanoreservoir chip also provides a way to investigate cell differentiation in the presence of multiple, spatially localized signaling agents for regenerative medicine. In addition to acting as a gate or valve for the reservoirs, the lipid bilayer gate could be utilized to present lipid-bound protein ligands or receptors. The reservoirs in the device can also be used as nano-reaction chambers, platforms for lipid studies, directors for self-assembly, and membrane-based protein detectors.

IV. Examples

The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention.

Example 1 Design and Fabrication of Exemplary Device

A device have an array of small reservoirs is prepared. Biological signaling agents are into each reservoir which is then sealed with a reversible lipid bilayer seal on top of a nanoscale pore in fluid communication with the underlying reservoir. The device is fabricated using focused ion beam or e-beam lithographic patterning and selective Si etching to create reservoirs beneath a SiO₂ membrane layer. Following patterning, a bilayer lipid membrane is deposited from a drug-containing solution via vesicle rupture onto the SiO₂ surface, trapping the drug within the reservoir. Example 2 Release of Agent from Device Reservoir

A device is prepared as described in Example 1, except the reservoirs are filled with a calcium-dependent fluorescent dye, Fluo-5N. The dye is placed in the reservoirs in a calcium-deficient nano-reservoir solution where the dye is not fluorescent. The amount and rate of dye released during electroporation of the lipid membrane gate is measured by electroporating the membrane for release of the dye into a high calcium medium (~5 mM). The quantity released is measured from the fluorescence intensity. The amount of dye released is varied by varying the voltage magnitude, pulse length, and pulse frequency of the electroporation. Based on the electroporation parameters established for Fluo-5N, the storage and release of cell-signaling molecules via fluorescence detection is evaluated. Specifically, release of the following agents is analyzed: fibroblast growth factor (FGF-2), bone morphogenesis protein-2 (BMP -2), retinoic acid, transforming growth factor βl (TGF- /31), and DNA oligomers. DNA electroporation is detected by electroporating into a solution of TOTO-I fluorophore (Molecular Probes, Eugene, Oregon) which is significantly brighter upon complexation with DNA, while the growth factors are detected by ELISA with their corresponding antibody (R&D systems, Minneapolis, MN).

Example 3: Release of Agents from Exemplary Device

A device is prepared as described in Example 1 , except the reservoirs are filled with FGF-2, BMP-2, or TGF-βl. Adipose-derived mesenchymal cells (AMCs) seeded and cultured on top of a nanoreservoir chip in control medium that is non-selective for differentiation along either the adipogenic and osteogenic pathway. The chip is arbitrarily sectioned for release of either adipogenic or osteogenic growth factor supplements from the reservoirs in the selected areas. Evidence for osteogenic differentiation is assessed by staining for alkaline phosphatase activity with Alizarin Red. Evidence for adipogenic pathway is detected by Oil Red O staining.

Briefly, the procedure is to make a 6 mg/mL solution of BPM-2 (for example, other molecules are similar); for 100 μg of BMP this is 16 μL of water. This solution is pipetted on top of the nanopore array, and the pores electroporated to store the BMP solution within the reservoirs beneath them. For one hundred 2 μm x 2 μm x 2 μm nano-reservoirs per electrode, a total volume of 0.8 nL can be contained. The BMP-2 solution is then rinsed off thoroughly with saline solution, and 50 μL of saline pipetted on top of the chip. The nanopores are then electroporated to release the BMP-2 into the pure saline solution. This supernatant solution is removed and analyzed with a BMP ELISA kit, per the manufacturer's instructions (R&D) Systems). Assuming 50% electroporation of the BMP solution into the supernatant, the concentration within the supernatant will be 100 pg/mL, which is clearly visible in the ELISA assay (cf. R&D Systems ELISA literature).

While the device and methods described above were illustrated as being used for cell processing, it should be understood that they can also be used for other applications, such as in a so called lab-on-a-chip (such as a fluid handling diagnostic device), an integrated microfluidics system (such as, for example, a fluid separation system for a chromatography or other fluid testing system), as well as in other sensors and drug delivery devices.

V. Device With Electrochemical Sealing and Opening of Small Opening

In another embodiment, an electrochemical seal and a method of forming the seal are described. The electrochemical seal is preferably a reversible barrier which may be used as the gate which seals and unseals a small opening, such as a nanopore, orifice, aperture, or a small channel, a plurality of times. For example, the electrochemical seal may be used as the gate which seals the nanopore to control the access of fluid to and from the reservoir shown in Figures 2C-2F. In other words, in one aspect, the electrochemical seal may be used instead of the lipid bilayer, flap, valve or bubble shown in the devices of Figures 2C- 2F. Thus, an electrochemical seal may be used to seal the nanopores in the nanopore array used for processing biological agents, such as cells, described with respect to the previous embodiments.

However, it should be understood that the electrochemical seal may be used in other applications, such as in a so called lab-on-a-chip (such as a fluid handling diagnostic device), an integrated microfluidics system (such as, for example, a fluid separation system for a chromatography or other fluid testing system), as well as in other sensors and drug delivery devices. In other words, the electrochemical seal may be used to seal any small opening in any device in which a fluid, such as a liquid and/or a gas passes through the opening. In this embodiment, an opening is considered "small" if it can be completely sealed or at least 50% sealed by electrochemical filling. Preferably, the opening has a width or diameter of less than 1 mm, such as 100 microns or less, such as 1 nm to 50 microns, including 25 nm to 1 micron.

Thus, in this embodiment, electrochemical deposition and dissolution may be used to seal and subsequently re-open small openings, such as to control fluid flow in a microchip. The opening and closing of a single opening or plurality of openings maybe individually controlled using digital or analog equipment such as a computer or a programmable voltage source.

The electrochemical sealing method includes depositing an electrochemically active species within a small opening, such as a small orifice or channel, to physically create a barrier between two initially communicating volumes. The process is illustrated in Figures 16A-16E. Initially the two volumes across the opening are in direct communication, and fluid, such as a liquid or a gas can flow or diffuse between the two. An active electrode is located within the opening or adjacent to the opening. The term adjacent to the opening means that the electrode is located sufficiently near to the opening such that by depositing electrochemically active species on the electrode at least partially seals the opening.

To seal the opening, the device is placed into a liquid solution or suspension. The solution of suspension may comprise: i) the same fluid that flows through the opening during the operation of the device; or ii) it may comprise the same fluid that flows through the opening during the operation of the device, but it which electroplatable metal ions are added prior to the voltage application to form the seal; or iii) it may comprise a special electroplating solution or suspension which is used only for sealing and unsealing the opening. Then, an appropriate voltage is applied between the active electrode and a counter-electrode elsewhere in liquid, reducing an electrochemically active species from solution and depositing the species onto the active electrode. After a sufficient amount of current flow, enough material is deposited so as to block the opening partially or entirely. The amount of progress during sealing can be monitored optically, such as by occlusion of light, or exclusion of a visible fluid from one side, or electronically, such as by monitoring the electrical resistance through the opening. Alternatively, the progress may be timed and the application of voltage is stopped after a predetermined amount of time which has been calculated to be sufficient to achieve a desired amount of sealing. The electroplating of material is stopped once a sufficient degree of sealing is achieved by turning off the voltage between the electrodes. The quality and functionality of the seal will depend on the type of material deposited. A wide range of active electrode materials can be used, such as metals and their alloys, such as Au, Ti, Pt, Fe, Ni, etc. or conductive organic materials, such as porphyrins, phthalocyanines, etc., each of which may have distinct characteristics and functionality. Any suitable conductive material which can be used for electroplating may be used. The electrochemically active species may comprise any suitable metal ions which can be plated from a solution, such as gold, nickel, copper and other metal ions. To reverse or remove the seal, the electrochemical potential is reversed leading to oxidation and dissolution of the deposited sealant material.

Figures 16A-16E illustrate metallic electrochemical sealing and unsealing of an opening 1 between the first volume 3 and a second volume 5. For example, the opening 1 may be a nanopore, the first volume 3 may be the volume above the nanopore and the second volume 5 may be the fluid reservoir. In the configuration shown in Figure 16 A, the active electrode 7 is located adjacent to the opening by surrounding a circular opening 1. The active electrode 7 is located between electrically insulating layers 9, 11. The bottom insulating layer 11 may be located on a substrate 13, such as a semiconductor (such as silicon or a compound semiconductor substrate, such as SiC, GaAs, GaN, etc.), glass, metal, plastic or ceramic substrate. If desired, initially the reservoir 5 may be filled with the solution to be stored.

As shown in Figure 16B, a metal 17, such as Au, is electrodeposited only upon the exposed active electrode 7 surface within the opening 1 upon the application of a voltage between the active electrode 7 and a counter-electrode 15 located in the same solution or suspension, hi other words, the metal is deposited on the sidewall of the active electrode 7 exposed in the opening. However, the metal is not deposited on the top and bottom surfaces of the active electrode 7 because they are covered by electrically insulating material layers, such as silicon oxide layers 9, 11. For example, a dilute solution of Au+ or other electroplatable metal ions may be used in the presence of the drug solution for the plating. Alternatively, the device may be moved between a first solution containing the metal ions for sealing the opening and a second solution for providing the drug / biological agent into the reservoir. The voltage is applied from any conventional voltage source 16, such as a battery and/or a grid powered voltage source. As shown in Figure 16C, after sufficient metal 17 deposition, the metal forms a seal 19 in the opening 1, trapping the drug inside the reservoir 5. The remainder of the electroplating solution and excess drug is washed away at this point. As shown in Figure 16D, the sealed reservoir 5 may be removed from solution for storage or handling.

As shown in Figure 16E, to remove the seal 19 from the opening 1 and to release the stored solution in the reservoir 5, the device is placed into a solution and the polarity of the electrochemical cell is reversed, causing the metal seal 19 to oxidize and dissolve back into solution. In other words, the polarity of the voltage between electrodes 7 and 15 is reversed compared to that in Figure 16B.

It should be noted that the insulating layers 9, 11 are optional. For example, if layer 9 is omitted, then this will merely result in an additional deposition of metal on top of the active electrode 7, which would be removed during the seal removal step shown in Figure 16E.

As an example, Figures 17A-17C illustrate the process for gold (Au) as an electroactive species 17. Initially the pore 1 is open, as shown in Figure 17A and a negative potential of -1.69 V (vs SCE) is applied to the active electrode 7 within the opening. Au⁺ or Au³⁺ within solution is reduced to form solid Au 17 on the electrode 7.

Further current will add more Au 17 to the electrode 7, eventually forming the seal 19 which closes the pore 1, as shown in Figure 17B. Once sealed, no further electrical stimulus is required to keep the pore 1 closed, and the pore can be stable for long periods. The device may be removed from the solution, but the Au seal 19 will remain.

The pore 1 is opened by reversing the potential to oxidize the deposited Au to Au+ once more, which subsequently diffuses away in solution, as shown in Figure 17C. As long as sufficient concentration of the electroactive species (such as Au+) is present at the electrode, this process can be repeated indefinitely or until material failure occurs.

As noted above, this device and method may be used in microfiuidic and biotechnology applications, especially as a way to seal select reagents into a chamber for long periods. In addition, it is of direct use for controlling drug delivery, such as that described above.

Figures 18A and 18B schematically illustrate two exemplary electrochemical sealing architectures. Figure 18A illustrates one arrangement for sealing a small pore 1 above a reservoir 5, such as in a microchip device describe above. Thus, Figure 18A is a three dimensional representation of the device shown in cross section in Figures 16A-E and 17A- C. The active electrode 7 contains an opening IA through it, which is aligned with the nanopore IB in the insulating layer 11. Alternatively, the active electrode 7 could be located only on one or more sides of the pore IB, without a hole IA extending through the electrode.

Figure 18B shows an alternative configuration where the active electrode 7 is used for forming a seal across a microfluidic channel 1C which is bounded by channel walls 19 and/or a substrate 13. In other words, the opening 1 in this configuration comprises the microfluidic channel 1C. The walls 19 may comprise any microfluidic wall material, such as a polymer material, semiconductor material (such as silicon) and/or an inorganic insulating material (such as silicon nitride). The substrate 13 may comprise any suitable substrate, such as a semiconductor, metal, glass, plastic or ceramic substrate. In this configuration, the active electrode 7 is located inside the opening 1 (i.e., inside the channel 1C). The active electrode 7 may or may not span the entire width of the channel 1C. In both cases a counter electrode is located elsewhere to complete the electronic circuit. Application of the appropriate voltage between the active electrode and a counter electrode in the presence of a redox active species will deposit this species within the channel 1C, eventually forming a seal 19 sealing the channel 1C to further fluid flow.

The device may also be used as a reversible wet fuse-antifuse, similar to the fuses and antifuses used in conventional semiconductor memory devices. In this case, the active electrode 7 shown in Figure 16A comprises two portions which are not connected to each other electrically. The metal seal forms an electrical contact between the two electrode 7 portions (i.e., forms a fuse or a "1" memory state). In contrast, when the metal βeal between the electrode 7 portions is removed, the electrical contact between the electrode portions is broken (i.e., it forms an anti-fuse of a "0" memory state). The voltage in a fuse / antifuse device may be reversibly applied a plurality of times to form a reversible wet fuse / antifuse. Also, the voltage may be applied between the counter electrode 15 and each separate portion of the active electrode 7 to enhance the fuse and antifuse formation. In this embodiment, the second volume, such as a reservoir 5, may be omitted and the electrode may be located directly on a substrate 13 or insulating layer 11.

Figure 18C shows an alternative configuration, in which the micro- or nano-pores are used together with a microfluidic system. In this configuration, a microfluidic channel 1C contains a barrier 11 containing one or more micro- or nano-pores 1. For example, the microfluidic channel 1C maybe located in a substrate 13, such as a semiconductor substrate, while the barrier 11 comprises a layer, such as a silicon oxide layer, containing the micro- or nano-pores 1. An electrode 7 electrochemically opens and seals the pores 1 by the method described above. This sealing and opening allows control of the fluid flow through and/or to and from the microfluidic channel 1C. Furthermore, if opening and sealing of different pores 1 or different sets of pores 1 are controlled by different electrodes 7, then it is possible to open and close access to the microfluidic channel 1C from different reagent reservoirs, vessels or channels 3A, 3B, 3C. Thus, the separate pores or sets of pores may be separately fluidly connected to different regent reservoirs, vessels or channels 3 A-C containing the same or different reagents. For example, the reservoirs may comprise reservoirs formed on the substrate either above or below the channel 1C, as described in the previous embodiments. The vessels or channels may comprise microfluidic vessels or channels which are formed on the same substrate as the channel 1C or which are located separately from the substrate. By selectively electrochemically opening and closing different pores or sets of pores 1, different reagents may be selectively provided to the channel 1C from one or more reservoirs, vessels or channels 3A-C and/or different reagents may be selectively provided from the channel 1C to particular reservoirs, vessels or channels 3A-C.

VI. Examples

The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention.

The device was fabricated using conventional semiconductor processing. An array of Pt electrodes was patterned onto an intrinsic silicon wafer with 100 nm of thermally grown oxide. A passivation layer is formed on top of the electrodes to electrically isolate the electrodes from the external solution. This can be any material which is electrically insulating, adheres well to the electrode, is robust enough to withstand extended periods in various aqueous environments, and inert enough to not cause interference with experimental systems (i.e. cell culture). The passivation layer for the exemplary device is a 1 μτa. layer of SU- 8 2 photoresist, spun-coat on top of the entire chip. Pores between 500 nm - 5 micron in diameter were then cut through the passivation layer, electrodes, and into the silicon wafer using a focused ion beam as shown in Figure 19 A. A silver wire is used as a counter electrode, and placed anywhere in the reduction and oxidation solution, but ideally as close to the pores as possible.

To seal the pores electrochemically, a negative potential of about -0.1 to -1.6 V is applied to the desired Pt electrode, depositing the Au⁺ or Au³⁺ ions from solution to form solid Au. This process is highlighted in Figure 19B. The reduction solution contains 0.025 M AuCl and 0.1 M KCl. By applying the voltage in a series of on-off pulses, the Au ions have time to diffuse between voltage pulses and allows for more control over the reduction process. If the 'on' pulses are short enough and the 'off recovery times long enough, then only the ions immediately around the electrode will be deposited during each pulse, ideally producing a nearly spherical gold seal which will plug, but not extend outside, the openings. For example, the off times may be 5-15 times as long as the on pulses and may be repeated for 50 to 500 cycles. In this example, 'on' pulses were 100 ms, and 'off recovery time was 900 ms and was repeated for 100 cycles. Upon completion of reduction and sealing of the reservoirs, the reduction solution is removed from the wafer while the solution beneath the Au plugs remains enclosed.

To electrochemically reverse the process a positive potential is applied to the Pt electrode, oxidizing the Au plug, as shown in Figure 19C. The presence of a small amount of chloride ions in solutions creates an electric potential region that thermodynamically favors formation of water-soluble chlorogold complexes, generally AuCl₄ ^", favoring more rapid dissolution. Most biological environments contain enough chloride ions to make cholo-assisted gold oxidation possible. It should be noted that many metals, such as Ni, do not require additional ions to help oxidize. The chip was placed into a Ix solution of phosphate-buffered saline (PBS) commonly used in cell culture, which contains approximately 0.145 M chloride ions at a physiological pH of 7.0. Figure 19C shows an SEM image of 4 pores after the Au plugs have been electrochemically dissolved at +1.1 V for 180 seconds. The metal has been completely removed from the pores, leaving the pores entirely open.

In summary, controlling fluid flow on very small (micron to nanometer) length scales is an area of great interest for developing lab-on-a-chip technology, integrated microfluidics, and drug delivery applications. A number of prior art methods of controlling fluid flow have been demonstrated, such as magneto-mechanical actuation, pneumatic compression, electrokinetic induced flow, and hydrogel swelling. However, many of these require continuous stimulation to remain in one particular state, and thus are not ideal for applications requiring long-term storage of a species, such as during storage and shipping. Secondly, many of these are relatively bulky and difficult to scale to small (<100 nm) length scales, or cannot be easily integrated into a vertical device.

In contrast, by using electrochemical deposition of the present embodiment avoids these problems by creating a permanent but reversible barrier to fluid flow that can be created or removed by application of an electrical potential (i.e., other words, the barrier is permanent in that it remains in the opening after the voltage is turned off- it does not require the voltage to be on continuously to remain either in the closed or open state). Since the barrier is electronically actuated, large arrays of these devices can easily be fabricated using traditional semiconductor techniques and controlled using computer electronics.

Although the invention has been described with respect to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

Claims

CLAIMS What is claim is:

1. A population of isolated cells cultivated on or over a semiconductor substrate comprising micro- and/or nano-scale reservoirs.

2. The population of cells as described above, wherein the cells are selected from the group consisting of stem cells, clonal stem cells, a substantially homogenous population of progenitor cells, a substantially homogenous population of expanded differentiated cells and a heterogenous population of progenitor cells and a heterogenous population of expanded differentiated cells.

3. The population of cells as described above, wherein the stem cells are embryonic stem cells or somatic stem cells.

4. The population of cells as described above, wherein the cells are actively propagating.

5. A method of cultivating isolated cells, comprising cultivating isolated cells on or over a semiconductor substrate comprising micro- and/or nano-scale reservoirs.

6. The method as described above, wherein the cells are stem cells.

7. The method as described above, wherein the stem cells are embryonic stem cells or somatic stem cells.

8. A substrate comprising one or more reservoirs having an average volume of less than about 10 pL and a gate or fluid regulating means for reversibly accessing the reservoirs.

9. A substrate comprising one or more reservoir having an average volume of less than about 10 pL, each reservoir being reversibly accessible by a pore having a width of less than about 10 μm.

10. The substrate as described above, further comprising a gate or a fluid regulating means disposed over the reservoir.

11. The substrate as described above, wherein the gate or the regulating means comprises at least one of a reversible membrane, such as a lipid membrane, a flap, a valve, a bubble or an electrochemically deposited seal.

12. The substrate as described above, wherein the substrate is comprised of: (a) a base composed of a first material and of a second material adjacent to the base; and (b) the reservoir being formed in one of the materials and the pore formed in the other material.

13. The substrate as described above, wherein an electrode is in contact with one or both of the materials to provide an electric field to or through the gate.

14. The substrate as described above, wherein the gate or regulating means comprises an active electrode located in or adjacent to the opening and a counter electrode.

15. The substrate as described above, wherein the active electrode is adapted to form at least a partial seal over the reservoir when the substrate is placed into a solution or a suspension and a voltage is applied between the active electrode and the counter electrode to electrochemically deposit a material from the solution or suspension in the reservoir.

16. The substrate as described above, wherein the active electrode is adapted to electrochemically remove the seal when the substrate is placed into a solution or a suspension and a opposite polarity voltage is applied between the active electrode and the counter electrode.

17. The substrate as described above, wherein the gate or regulating means is closed over the reservoir.

18. The substrate as described above, wherein the reservoir further comprises a liquid or a fluid composition.

19. The substrate as described above, wherein the composition further comprises an agent selected from the group consisting of a growth medium, a biologically active agent, and a pharmaceutical composition.

20. The substrate as described above, wherein the composition comprises a cellular growth medium.

21. The substrate as described above, wherein the cellular growth medium comprises a cell differentiation medium.

22. The substrate as described above, wherein the cell is an isolated stem cell.

23. The substrate as described above, wherein the isolated stem cell is an embryonic stem cell or a somatic stem cell.

24. The substrate as described above, wherein the cell differentiation medium is selected from the group consisting of adipogenic differentiation medium, osteogenic differentiation medium, leiomyogenic differentiation medium, chondrogenic differentiation medium, myogenic differentiation medium and neuronal differentiation medium.

25. A substrate comprising: (a) one or more micro- or nano-reservoir(s) containing an average volume of less than about 10 pL of cellular growth medium, each reservoir being reversibly accessible by a nanopore having a diameter of less than about 10 μm; and (b) an isolated stem cell.

26. A substrate comprising: (a) one or more micro- or nano-reservoir(s) containing an average volume of less than about 10 pL of cellular growth medium, each reservoir being reversibly accessible by a nanopore having a diameter of less than about 10 μm; and (b) a population of stem cells.

27. The substrate as described above, wherein the population of stem cells is selected from the group consisting of clonal stem cells, a substantially homogenous population of progenitor cells, a substantially homogenous population of expanded differentiated cells, a heterogenous population of progenitor cells and a heterogenous populaton of expanded differentiated cells.

28. A substrate comprising: (a) one or more micro- or nano-reservoir(s) containing an average volume of less than about 10 pL of cell differentiation medium, each reservoir being reversibly accessible by a nanopore having a diameter of less than about 10 μm; and (b) a heterogenous population of cells expanded from an isolated stem cell.

29. A device, comprising one or more micro- or nano-reservoir(s), each reservoir being accessible by an aperture, such as a nanopore.

30. The device as described above, further comprising a gate or a fluid regulating means disposed over the nanopore.

31. The device as described above, wherein the gate or the regulating means comprises at least one of a reversible membrane, such as a lipid membrane, a flap, a valve, a bubble or an electrochemically deposited seal.

32. The device as described above, wherein the reservoirs are loaded with one or more reactants or agents, such as stem cells, for release in response to a stimulus.

33. The device as described above, wherein: the device is comprised of a base composed of a first material and of a second material adjacent to the base; and the reservoir(s) being formed in one of the materials and the nanopore formed in the other material.

34. The device as described above, wherein electrodes are in contact with one or both of the materials to provide an electric field to or through the gate.

35. A device, comprising: an opening located between two communicating volumes; an active electrode located in or adjacent to the opening; and a counter electrode; wherein the active electrode is adapted to form at least a partial seal in the opening when the device is placed into a solution or a suspension and a voltage is applied between the active electrode and the counter electrode to electrochemically deposit a material from the solution or suspension in the opening.

36. The device as described above, wherein the active electrode is adapted to electrochemically remove the seal when the device is placed into a solution or a suspension and a opposite polarity voltage is applied between the active electrode and the counter electrode.

37. A device, comprising: an opening located between two communicating volumes; a counter electrode; a first means for forming at least a partial seal in the opening when the device is placed into a solution or a suspension and a voltage is applied between the first means and the counter electrode to electrochemically deposit a material from the solution or the suspension in the opening.

38. The device as described above, wherein the first means is also a means for electrochemically removing the seal when the device is placed into a solution or a suspension and a voltage is applied between the first means and the counter electrode.

39. A device, comprising: an opening located between two communicating volumes; an active electrode located in or adjacent to the opening; a counter electrode; and a first means for applying a voltage between the active electrode and the counter electrode to electrochemically deposit a material from a solution or suspension in the opening to at least partially seal the opening.

40. The device as described above, wherein the first means is also a means for applying an opposite polarity voltage between the active electrode and the counter electrode to electrochemically remove the seal when the device is placed into a solution or a suspension.

41. A device, comprising: an opening located between two communicating volumes; a seal located in the opening; an active electrode located in or adjacent to the opening; and a counter electrode; wherein the active electrode is adapted to electrochemically remove the seal when the device is placed into a solution or a suspension and a voltage is applied between the active electrode and the counter electrode.

42. A device, comprising: an opening located between two communicating volumes; a seal located in the opening; a counter electrode; and a first means for electrochemically removing the seal when the device is placed into a solution or a suspension and a voltage is applied between the first means and the counter electrode.

43. A device, comprising: an opening located between two communicating volumes; a seal located in the opening; an active electrode located in or adjacent to the opening; a counter electrode; and a first means for applying a voltage between the active electrode and the counter electrode to electrochemically remove the seal when the device is placed into a solution or a suspension.

44. A method for expanding an isolated cell comprising contacting the cell with a substrate or device as described above under conditions suitable for the expansion of the cell thereby expanding the cell.

45. The method as described above, wherein the cell is selected from the group consisting of an embryonic stem cell, a somatic stem cell and a progenitor cell.

46. A method for preparing a pre-determined tissue composition comprising contacting an isolated stem or progenitor cell with a substrate or device as described above under conditions suitable for the expansion of the stem or progenitor cell into the pre-determined tissue composition.

47. The method as described above, wherein the suitable conditions comprise contacting the cell with an effective amount of a cellular growth medium.

48. The method as described above, wherein the cellular growth medium is a stem cell differentiation medium.

49. An in vitro method to assay for modulation of the expansion of an isolated stem cell or an isolated population of stem cells, comprising contacting the isolated stem cell or the isolated population of stem cells on the semiconductor substrate as described above and in the presence of a test agent and monitoring the effect of the test agent on the expansion of the isolated stem cell or the isolated population of stem cells.

50. An electrochemical sealing method, comprising: placing a device into a solution or a suspension, wherein the device comprises an opening located between two communicating volumes, an active electrode located in or adjacent to the opening, and a counter electrode; and applying a voltage between the active electrode and the counter electrode to deposit a material from the solution or the suspension in the opening to at least partially seal the opening.

51. The sealing method as described above, further comprising unsealing the opening by applying a voltage of an opposite polarity between the active electrode and the counter electrode to remove the deposited material from the opening.

52. An electrochemical unsealing method, comprising: placing a device into a solution or suspension, wherein the device comprises at least a partial seal located in an opening which is located between two communicating volumes, an active electrode located in or adjacent to the opening, and a counter electrode; and applying a voltage between the active electrode and the counter electrode to remove the seal from the opening.