WO2024006673A1

WO2024006673A1 - Stem cell and organ maturation system and method

Info

Publication number: WO2024006673A1
Application number: PCT/US2023/068996
Authority: WO
Inventors: Luiz C.N. SAMPAIO; Doris A. Taylor
Original assignee: Regenmedix Llc
Priority date: 2022-06-30
Filing date: 2023-06-23
Publication date: 2024-01-04

Abstract

A system and method for maturing reliable cardiac cells and organs which employs an electro-mechanical approach which mimics cardiac physiology, both regionally and temporally. The system employs local biochemical cues, electrical cues, and mechanical cues which synergize to provide conditions similar to natural cell development in order to yield superior stem cell maturation to adult phenotypes. A microcontroller is disposed in communication with a 3D heart scaffold, an aortic (natural orifice) pump, and a left atrium pump to provide the requisite cues for maturation. A specially-designed liquid medium supports the immature cells within the heart scaffold until their maturation. The system is applicable to the maturation of other organs beyond that of the heart.

Description

STEM CELL AND ORGAN MATURATION SYSTEM AND METHOD

CONTINUITY

This application is a non-provisional application of provisional patent application number 63/357,262, filed on June 30, 2022, and priority is claimed thereto.

FIELD OF THE PRESENT INVENTION

The present invention relates generally to stem cell and/or organ maturation technology, and more specifically relates to a method and tunable system to drive cell and/or organ maturation. Further, the present invention embodies a system facilitating pluripotent or multipotent stem cell maturation via an organ scaffold preferably disposed in communication with an electrical signal and at least one device, facilitating electrical and/or mechanical stimulation of the cells.

BACKGROUND OF THE PRESENT INVENTION

Induced Pluripotent Stem Cells (iPSCs) are complicated and can be hard to make for a specific type of mature, functional cell. Historically, iPSCs have been made by transforming one type of cell with a particular function into a new type of cell with a different function. However, when producing iPSCs for cardiac tissue, the cells fail to function as mature cells. These cardiac cells are not mature enough to accurately demonstrate in vitro or in vivo functions associated with traditional mature (adult) cardiac cells. Therefore, drug testing on these cardiac cells is incomplete or inconclusive because the cells fail to accurately represent infant and/or adult heart or cardiac functions.

Human embryonic stem cell (hESC) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), as they are currently derived and differentiated, are electrically and mechanically immature compared to neonatal adolescent or adult heart cells. Because of this immaturity, the cells fail to adequately predict adult heart responses to stimuli, including, but not limited to drugs. Because of this immaturity, the cells fail to safely integrate into an adult heart, causing electrical or mechanical instability and/or tumors. Because of this immaturity, the cells fail to generate enough contractile force to adequately power a tissue-engineered cardiac construct, such as either a patch, a valve, or a whole heart.

There are several existing techniques to solve this problem, all techniques lacking effectiveness and reliability. A first approach to mitigate electrical instability of hiPSC-CMs involves gene editing of the cells to express adult cardiac cell ion channels. This both requires cell-type specific editing and, more broadly, can lead to genetic instability of reprogrammed cells. A second approach is to generate a three-dimensional electro-spun cardiac testbed meant to simulate a ventricle testbed. This approach lacks vasculature and testbed suffers from low reliability, low viability, and only the potential to generate ventricular cells. A third approach is a two-dimensional scaffold subject to intermittent linear or axial stretching, which fails to generate reliable mature cells. A final approach is substrate stiffness-induced maturation, subjecting immature stem cell derived cardiac cells to increasing stiffness against which they contract to improve force generation over time, but again lacks reliability upon maturation.

Therefore, there exists a need for developing stem cell maturation to adult phenotypes. There exists a need for developing stem cell maturation, generating stem cells usable for drug testing and other uses.

SUMMARY OF THE PRESENT INVENTION

The present invention is a system configured to yield superior stem cell maturation to adult phenotypes and/or to facilitate organ maturation. The system employs an electromechanical approach that mimics cardiac physiology both regionally and temporally. Further, the system of the present invention provides nascent cells both local biochemical cues (within the scaffold matrix of the present invention), electrical cues (from the stimulation provided by the system of the present invention), and mechanical cues (from pumps that provide pressure and fluid flow combined with scaffold biomechanics) that synergize to provide conditions similar to natural cell and organ development. The electrical and mechanical components employed in the system of the present invention are tunable such that the electrical and/or mechanical cues asserted need not be fixed. Cues are configured to be issued that are pertinent for the cells of an individual, and then the cues may be increased or decreased as the cells and/or organs mature. The following brief and detailed descriptions of the drawings are provided to explain possible embodiments of the present invention but are not provided to limit the scope of the present invention as expressed herein this summary section.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.

The present invention will be better understood with reference to the appended drawing sheets, wherein:

FIG. 1 details the four-stage process of the present invention.

FIG. 2 exhibits a flow chart detailing the process of use of the apparatus of the present invention by a user.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present specification discloses one or more embodiments that incorporate the features of the invention. The disclosed embodiment(s) merely exemplify the invention. The scope of the invention is not limited to the disclosed embodiment(s). References in the specification to “one embodiment,” “an embodiment,” “an example embodiment,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment, Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

The method and system of the present invention is a physiological electro-mechanical approach, combined with cells located in an appropriate environment, for creating human cardiac cell maturation from pluripotent or multipotent human cells created in vivo or in vitro (one embodiment is hESC- or hiPSC-CM). Likewise, the system and method of the present invention can be employed to facilitate the maturation of gene-edited cells, as well as other human stem cells capable of originating cardiomyocytes, including, but not limited to cardiac-derived, bone marrow-derived, blood-derived, and adipose tissue-derived cells.

The method and system require placing cells in a stimulative environment and driving the cells to work over a dynamic range of activities that mimics the increasing work occurring during organ (for example, the heart) development. The method and system expose cells to a cardiac (or other applicable organ) extracellular environment of an appropriate type combined with the application of electrical and mechanical stimulation to provide multiple cardiomyocyte (or other mature cell) lineages (regional specific parenchymal cell types) and other vascular and parenchymal cell phenotypes.

Fig. 1 illustrates one embodiment of the system providing for cell maturation to adult phenotypes. The system employs a three-dimensional (3D) heart scaffolding structure. In one embodiment, the heart scaffolding structure may be similar to the scaffolding structure described in the Handbook of Tissue Engineering Scaffolds: Volume one, Number 29 - Whole-heart scaffolds - how to build a heart available from Woodhead Publishing Series in Biomaterials, 2019, pages 617-642. The 3D heart scaffolding structure includes 2 or more chambers of the heart, including by way of example but not expressly limited to right chambers, left chambers, both ventricles, both atria, great vessels, etc.

The method and system additionally employ one or more pumps which are configured to provide pressure and cause a liquid/medium to flow. Herein, the pumps may be any suitable pump operative to perform operations in response to microcontroller operations. In one embodiment, the pump may be a CE ROHS High Flow Peristaltic Pump Stepper Motor 24v with Quick Tubing Load Pump Head YZ2515.

The pumps operate to pump a medium into the 3D heart scaffolding structure. The sequencing of pump operations mimics heartbeats. The pumps can operate in accordance with known pump operations, pumping the liquid / medium for defined time periods and/or until a pressure value or range is achieved, as well as controlling the direction of the liquid / medium flow. The microcontroller is preferably configured to provide a pump start instruction, a pump flow direction instruction, a pump volume or force instruction. The medium employed in some embodiments of the present invention is preferably a proprietary mixture that enables the survival and maturation of both vascular and cardiac parenchymal cells. Therefore, the medium enables survival and maturation of both compartments, and does not sacrifice one for the other.

The medium employed in the system of the present invention can be any suitable liquid as recognized by one skilled in the art. In one embodiment, the medium is cell culture media or a liquid having the viscosity and other flow parameters mimicking cell culture media. In another embodiment the medium is blood, such as donated human blood. In another embodiment, the medium can mimic or simulate blood by including packed red cells or cells derived from blood. In another embodiment, the medium is a liquid having viscosity and other flow parameters mimicking blood flow. In another embodiment, the medium is a liquid having oxygen delivery capabilities. The above examples are exemplary and not an exclusive or exhaustive list.

The pumps are controlled by a microcontroller. The microcontroller may be any suitable type of processing device or devices measuring feedback parameters and generating control or engagement instructions to connective elements. As noted in further detail below, the microcontroller generates pump instructions to the different pumps, as well as electrical pulses to the 3D heart scaffolding structure.

The system can include a pacemaker receiving electrical charge instructions from the microcontroller. The pacemaker provides a timed electrical charge at an instructed power level

(milliamps) to the cells in the 2D or 3D heart scaffolding structure. In one embodiment, the electrical pulses and/or patterns synchronize as the heart cells mature. Therefore, as described in greater detail below, the strength of the electrical charge increases as the cells mature.

The present method and system operate, in one embodiment, on a four-step continuous sequence of operations. Fig. 2 illustrates a method flowchart of sequential steps, with further illustration of timing sequence is noted in Fig. 1.

In the flowchart, step 200 is loading immature cells into the 3D heart scaffolding. In one embodiment, the immature cells are iPSCs. Additional cell types may be utilized herein, including but not limited to, other cells differentiated down cardiac lineage includes embryonic stem cells (hESC) or potential mesenchymal stem cells, or in another embodiment immature heart cells derived from humans. In one embodiment, the cells can be part of a cell culture system.

As the immature cells may be of a variety of cell types, varying in developmental lineage, the system of the present invention employs a process designed to mature the organ, which, by requirement, means maturing the cells present to enable whole organ function. The cells delivered to the organ parenchyma include a mixture of cells differentiated to cardiac mesodermal cells and a subset, then, to cardiomyocytes of various types. The delivered cells are distributed ubiquitously and are then driven to differentiate and functionally mature by the systemic biochemical electrical and mechanical cues provided. The overall cues mimic a cardiac cycle as indicated above - allowing the cells to receive temporally correct and mechanically “correct” cues in the face of appropriate biochemistry and ECM enabling maturation at a level not previously available for a cardiac system ex vivo/in vitro. The signals are designed to capture the stimulation of the cardiocytes as a whole in a manner that can be recorded. Regional stimulation can occur by stimulating single leads or regions of applied electrodes.

In the current embodiment, the electrodes are a single wire placed locally. However, this need not be employed in all designs of the application of the system and method of the present invention. The final design may include a mesh or soft electrically active substrate which is applied to the surface of the heart to enable stimulation more globally with single signals possible at each mesh point. Stimulation is applied at a single or multiple points via electrically conductive material (such as electrode wires, nano wires, and/or electrically active gels) until depolarization of the cell membrane occurs and can be recorded. When this is recorded from the cells present, it indicates electrical capture and provides a starting point for stimulation of that heart. This facilitates personalized tunability of the system in accordance with the needs of the cells being stimulated..

Once loaded, the below steps are iterative operations for progressing the cell maturation.

It is recognized the steps are iterative steps, the first iteration being for beginning the maturation process and that the below-noted sequence of step 202 as the first step is not an express sequential requirement. Step 202 is filling the left ventricle with a controlled volume of liquid, causing a controlled stretch of the cells in the scaffolding. In this embodiment, the left atrium pump receives a pump instruction from the microcontroller to pump the medium into the left ventricle chamber of the 3D heart scaffolding.

Illustrated in Fig. 1, the left atrium pump fdls the left atrium in response to the microcontroller commands. The left atrium pump fills the left atrium and then into the left ventricle, causing a controlled stretch of the cells loaded on to the 3D heart scaffold.

In one embodiment, the system includes a plurality of pressure transducers measuring the pressure values of the liquid being pumped by the left atrium pump and/or the aortic pump. These pressure transducers generate pressure readings, which are received by the microcontroller. Therein, the microcontroller can then modify the pump instructions to the pumps based on the pressure readings from the pressure transducers.

In the clock illustration of Fig. 1, this is step 2, the second part of the diastolic phase.

Once the cells are properly and/or effectively stretched from the pressure on the scaffolding from the left atrium pump, step 204 is triggering an electrical pulse from the microcontroller to the cells loaded into the 3D heart scaffold. The electrical pulse causes the membrane of the cells to depolarize leading to contraction. This electrical-induced contraction therefore mimics the isovolumetric phase of a regular heartbeat cycle. Tn Fig. 1 , this is illustrated as step 3, application of the pacemaker pulse. As the cells mature, the amount of electrical charge may be increased or decreased. For example, as the cells mature, they grow in size and strength. As the 3D heart scaffolding increase in area (i.e. with a larger heart), the higher electrical charge may be needed to counteract the increased size and produce the same isovolumetric contraction. In one exemplary embodiment, the range of electrical charge can range from a low end of 3mA to an upper range of 20 mA.

It is further within the scope herein that the electrical charge can vary based on the cell types or volume of cells placed in the 3D heart scaffolding. For example, a larger cell number or higher density may require a greater electrical charge than a smaller cell number/density. Herein, the present invention can operate using varying techniques, including a trial and error of varying electrical charges and measuring isovolumetric contraction as part of the systolic phase or in another embodiment processing calculations can correlate the electrical charge with the cell type, cell volume, and cell maturity.

To continue with the stimulated heart function, step 206 is reducing the ventricular pressure with an aortic pump. In Fig. 1, the aortic pump withdraws fluid or medium from the aortic chamber of the 3D heart scaffolding. This reduction of fluid or medium occurs for a defined period of time to complete the systolic phase of the simulated heartbeat.

In one embodiment, a pressure sensor monitors the aortic pump flow pressure to determine the appropriate ventricular pressure reduction. As noted above, pressure transducers measure the pressure of the liquid / medium. This value can be translated to represent the pressure at the aortic valve of the 3D heart scaffolding. Tn one embodiment, the operational ranges for aortic pressure are between 30 and 150 mm Hg, whereas the pressures can be higher and/or lower based on operational conditions and the above range is not an express limitation.

Once the ventricular pressure reduction is determined, step 208 is reversing the aortic pump direction and re-establishing aortic pressure for coronary perfusion. This step 208 is beginning of a next diastolic phase.

By reversing the aortic pump, the 3D heart scaffolding reverts the aortic pressure for the diastolic phase. The aortic pump fills the aorta of the 3D heart scaffolding to a target pressure. In one embodiment, the operational ranges for aortic pressure are between 30 and 150 mmHg, whereas the pressures can be higher and/or lower based on operational conditions and the above range is not an express limitation.

In the event the cells are not deemed matured, step 210, the method reverts back to step 202. Based on the microcontroller instructions, the left atrium pump fdls the left atrium with the controlled volume of liquid. This liquid volume increase expands the 3D heart scaffolding, causing the cells to expand.

The steps 202-208 are iterated in a loop, simulating heartbeat movements and the microcontroller injecting electrical charges to simulate physiological stimulation. As shown in Fig. 1, the process of steps 1-4 (steps 208, 202, 204, and 206) continue in an iterative loop. As the cells mature, the values of the iterative loop dynamically adjust. Maturing cells may require greater aortic pressure, may require a higher electrical pulse, and may require greater ventricular pressures. The adjustment of these values and instructions from the microcontroller can vary based not just on the type of cell originally placed on the 3D heart scaffolding, but also the volume or number of cells placed on the 3D heart scaffolding. Moreover, all cells have unique properties and therefore even identical cell types can have unique maturation rates. Therefore, the microcontroller operates using a dynamic range of instructions to the pumps and pacemaker.

Initially the electrical stimulation provided by the system of the present invention is designed to provide a field effect because the cardiac cells have not yet connected to form an electrical syncytium. Over time, as the heterogenous cells present proliferate, align (relative to the matrix and load), and connect via gap junctions, an electrical syncytium is formed. Then, the electrical stimulation is conducted from one cell to another, and its efficacy to depolarize the cells will depend on multiple factors like distribution and type of the cells in the scaffold, size and number of the cells, and impedance of the myocardial wall to electrical propagation. As the cardiomyocytes mature, the size and mass of the cells increase. As myocardial mass increases, electrical impedance may increase, requiring an increase in the nominal electrical stimulation provided by the system of the present invention.

The microcontroller of the present invention is key to controlling both the electrical and mechanical input in a temporally coordinated manner that mimics the cardiac cycle in both duration and volume. The microcontroller is preferably equipped with as many electrical circuits as necessary to handle a minimum of two pumps, as described in Fig. 1 . The addition of pumps to control flow regionally can be imagined under conditions where a valve, vessel, chamber, or wall region require specific inputs for a period of time. Each electrical stimulus equates to a cardiac beat-per-minute and generates a cardiac cycle. Targeted stimulation parameters are based on achieving a final heart rate of 70 ±10 beats per minute. The initial rate is designed to mimic fetal heart rate (110-160 bpm). Progression to a nominal rate of 70+- 10 bpm is pursued as the cells are matured. The flow of the pumps is calculated based on the size of the 3-D scaffold and the heart rate to achieve sufficient load on the chambers to mimic developmental pressures.

Duration of exposure to the system depends on the level of functional maturation of the heart achieved over time. When the heart, in total, has an EF> 50%, duration is evaluated for completion. Electrical maturation of individual cells is not necessarily measured in the system. However, in a parallel plate of cells, electrical maturation may be quantified in a subset of hearts as a quality indicator.

In one embodiment, the microcontroller employs processing logic for receiving and processing incoming pressure signals from the pressure transducers disposed between the pumps and the 3D heart scaffolding. The pressure transducers can measure contractile force of the heart based on the pressure of the medium and knowing the pump force output values. The pressure transducers to be used are preferably similar to clinically-approved commercially available transducers. Tn another embodiment, the cell maturation can be determined based on drug response.

Therefore, external drug applications may be performed and measured, with feedback given to the microcontroller through a user interface. The microcontroller can therefore assess the maturation stage and maintain or modify the output instructions to the pumps and pacemaker.

In one embodiment, the perfusion-stimulation loop and microcontroller instructions to the pumps and pacemaker can vary based on the maturation phase of the cells. For instance, in the initial phase of maturation, the microcontroller may include additional feedback loops or modifications to ensure the cell maturation process initiates. Then, once maturation has begun or reached a specific stage or rate of maturation, the microcontroller may operate on a more automated phase.

As the perfusion-stimulation loop iterates, the cells deposited on the heart scaffolding reach maturity. Therefore, the 3D heart scaffolding now holds a functioning heart with mature and functioning heart cells. It should be noted that the size of the scaffold, which will be chosen in dependence of the number of cells to be matured or size of the organ required, will determine the volumes to be pumped. The electrical stimulation is preferably calibrated at the starting point of the process. Electrical signals that are low and fail to capture the cardiomyocytes present in the scaffold will fail. Signals that are too strong for the cells will damage or kill the cells and fail. So, finding the correct starting point for each heart is key. The optimal starting point may be determined in a dish of cells disposed in parallel to those of the scaffold, or typically the process employs a very low signal current to begin in order to achieve coordination of the initial cell depolarization alter the pulse as the low starting point. Therefore, a final step in the process is the harvesting of the generated heart, step 210. This harvesting can include, for example, being transplanted into a human. The system of the present invention may permit the transport of a heart under living conditions such that stimulation is continued via the use of a separate stimulation perfusion apparatus in future applications.

In contrast to the preexisting cell maturation methods cited in the prior art, the superiority of the method and system of the present invention lies in the fact that cardiomyocytes need both electrical and mechanical stimulation to contract and develop. As early as in the tubular heart, the most primitive mesodermal-derived structure, the cardiomyocytes are under electrical stimulation and mechanical stress. As soon as the initiation of heartbeat, the cardiac tube undergoes a process of looping, which leads to the period of chamber formation and then blood flow. Starting electrical and mechanical stimulation as early as possible alter the cell commitment to the cardiomyocyte lineage and will provide the environmental cues necessary for their maturation. The system of the present invention combines all important cues for cell development and maturation; macro, micro, and nano structures provided by the 3-D scaffolds where the cells are placed in electrical and mechanical (load/unload) conditions provided by the timed coordination of stimulation and fluid flow via pumps in a way that mimics the ventricular cycles.

It should be noted that the present invention is a system comprised of physiologic perfusion and mechanical stretching and/or compression of cells, and with, or without, electrical stimulation that, when applied to immature cells in a 2D or 3D organ scaffold under appropriate conditions, can drive cell organ component and whole organ maturation effectively and efficiently to yield stable mature cells and/or organs. Further, the present invention is a system designed to mature the parenchymal cells in an organ scaffold in a manner that leads to the functional maturation of that organ. One application, or embodiment, of the present invention is for the maturation of stem or progenitor cells placed into the parenchyma of a heart as described. Further, another application of the system of the present invention is to mature cells such that they may be used for drug testbeds, cell therapy products, and other research or medical applications.

It should be understood that all embodiments of the system and method of the present invention need not employ a pump-based mode, but all embodiments employ a perfiision/flow system or mechanism to create or generate physiologic pressures and appropriate mechanical compression/distension and employs electrical stimulation at appropriate times individually or with mechanical cues to promote cell proliferation, cell stimulation, and/or cell depolarization when appropriate. When the system of the present invention is applied to cells in an organ scaffold, the system drives cell maturation as well as organ maturation. The system of the present invention is a necessary component of organ maturation ex vivo.

The included figures are conceptual illustrations allowing for an explanation of the present invention. Notably, the figures and examples above are not meant to limit the scope of the present invention to a single embodiment, as other embodiments are possible by way of interchange of some or all of the described or illustrated elements. Moreover, where certain elements of the present invention can be partially or fully implemented using known components, only those portions of such known components that are necessary for an understanding of the present invention are described, and detailed descriptions of other portions of such known components are omitted so as not to obscure the invention. In the present specification, an embodiment showing a singular component should not necessarily be limited to other embodiments including a plurality of the same component, and vice-versa, unless explicitly stated otherwise herein. Further, the present invention encompasses present and future known equivalents to the known components referred to herein by way of illustration.

Having illustrated the present invention, it should be understood that various adjustments and versions might be implemented without venturing away from the essence of the present invention. Further, it should be understood that the present invention is not solely limited to the invention as described in the embodiments above, but further comprises any and all embodiments within the scope of this application.

The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiment was chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.

Claims

CLAIMS We claim:

1. A system configured to reliably and efficiently mature living cells and organs comprising: loading immature cells into an organ-derived scaffold; a regional pump receives a pump instruction from a microcontroller; the regional pump pumping a liquid medium into a region/lobe/area of the organ scaffold causing a controlled stretch of the cells, constituting a perfusion of the cells of a perfusion-stimulation loop; the microcontroller triggering an electrical pulse; the electrical pulse causing the cells to contract , mimicking an isotonic stretch phase of a natural organ, (or isovolumetric in the case of the heart), constituting stimulation of the cells of the perfusion-stimulation loop; reducing regional pressure via a natural orifice pump; reversing the direction of the orifice pump; and reestablishing orifice pressure for organ perfusion during a relaxation phase, thereby continuing the perfusion-stimulation loop.

2. The system of claim 1, further comprising: monitoring the development of the immature cells over time during repeated instances of the perfusion-stimulation loop; and the microcontroller adjusting the current applied in the electrical pulse in accordance with the maturity of the cells. The system of claim 1, further comprising: the cells developing to maturity; and transplanting the cells. The system of claim 1, wherein the microcontroller is disposed in communication with a power source; and wherein the microcontroller is disposed in communication with the organ scaffold via electrodes. The system of claim 1, wherein the organ scaffold is two dimensional. The system of claim 1, wherein the organ scaffold is three dimensional. The system of claim 2, further comprising: the cells developing to maturity; and transplanting the mature cells. The system of claim 3, wherein the microcontroller is configured to regulate the rate at which the at least one pump; and wherein the microcontroller is tunable such that it is configured to regulate the current applied to the organ scaffold between the range of 0.3mA and 20mA. The system of claim 3, wherein the volume of the medium is tunable in proportion to the number of cells to be matured. The system of claim 3, wherein the operational ranges for natural orifice pressure as applied by the at least one pump are between 10 and 150 mm Hg. A system configured to reliably and efficiently mature living cells comprising: first, loading immature cells into an organ scaffold; second, a regional pump receives a pump instruction from a microcontroller; third, the regional pump pumping a liquid medium into the region/lobe of the organ scaffold causing a controlled stretch of the cells; fourth, the microcontroller triggering an electrical pulse; fifth, the electrical pulse causing the cells to contract, mimicking an isotonic contraction phase of a natural organ or isovolumentric of the heart; sixth, reducing regional pressure via a natural orifice pump; seventh, reversing the direction of the orifice pump; and eighth, reestablishing aortic pressure for parenchymal during a relaxation phase. The system of claim 9, further comprising; monitoring the development of the immature cells over time; and the microcontroller adjusting the current applied in the electrical pulse in accordance with the maturity of the cells; the cells developing to maturity; and transplanting the cells. The system of claim 10, wherein a size of the organ scaffold is selected in dependence of the number of cells to be matured; and wherein a volume of the medium to be pumped is determined by the number of cells to be matured. A system configured to reliably and efficiently mature living cells and/or organs comprising; loading immature cells into a three-dimensional organ scaffold; exposing the immature cells to a liquid medium; the liquid medium disposed in a state of intermittent oscillating flow, supplying a pressure to the immature cells; exposing the immature cells to an electrical current; a microcontroller regulating the amperage of the electrical current; the microcontroller regulating the rate of the intermittent oscillating flow of the liquid medium; and the electrical current combined with the intermittent oscillating flow of the liquid medium generating physiologic pressures to the immature cells, appropriate mechanical compression/distension of the immature cells, and electrical stimulation to the immature cells, promoting cell proliferation, cell stimulation, and cell depolarization to drive cell maturation, creating mature cells and facilitating organ maturation.