WO1999023199A1

WO1999023199A1 - Closed system for processing primary tissue

Info

Publication number: WO1999023199A1
Application number: PCT/US1998/022563
Authority: WO
Inventors: Mark B. Jones; David A. Maryanov; Robin L. Geller; Steven K. Neuenfeldt
Original assignee: Baxter International Inc.
Priority date: 1997-10-31
Filing date: 1998-10-26
Publication date: 1999-05-14

Abstract

The present invention relates to methods and kits for tumor processing in a closed environment, using blunt dissection by solid particles. The tissue dissociation time is significantly less than that needed for conventional methods (minutes versus hours). The methods and kit of the invention allow greater surface areas of tumor to be dissociated at any one time due to the increased bead/tumor contact surface area. Primary tumors were reproducibly dissociated into viable heterogeneous single cell suspensions using the methods and kit of the invention, while retaining cell function.

Description

CLOSED SYSTEM FOR PROCESSING PRIMARY TISSUE

Field of the Invention

This invention relates to kits and methods for dissociating primary tumors and other tissues into viable single cell suspensions while maintaining an aseptic environment.

Background of the Invention

Cells which secrete therapeutically useful substances can be used in a number of applications, including gene therapy for treating cancer and other diseases. The isolation of tumors and other tissues provides a source of cells for genetic engineering and, subsequently, for gene therapy purposes. However, methods to effectively dissociate the isolated tissue mass into viable single cells or cell clusters are necessary in order to utilize these cell sources. Processing of primary tumor biopsies to yield heterogeneous single cell suspensions currently takes several hours and usually requires enzymatic chemical digestion and shear mechanical dissociation (Freshney R.I. Disaggregation of the Tissue and Primary Culture. Chapter 11 in Culture of Animal Cells A Manual of Basic Technique, 99-118. New York: Alan R. Liss, Inc., 1983). These methods tend to damage the cells due to the slow rate of dissociation. Furthermore, conventional methods are prone to contamination because aseptic technique is extremely difficult to maintain. Accordingly, there is a need in the art for an improved method for preparing viable single cell suspensions or clusters of cells from solid tissue samples while maintaining an aseptic environment. Summary of the Invention

The present invention provides an improved method and a kit for preparing

viable single cell suspensions or clusters of cells from solid tissue samples while

maintaining an aseptic environment. An aseptic environment is maintained by

dissociating tissue samples in a closed environment. As used herein, the term "tissue

samples" includes both tumorigenic and nontumorigenic tissues. As used herein, the

term "closed environment" refers to a container that is closed once the tissue sample is

placed in the system. The mechanical dissociation utilized in the present method also

allows for more uniform and rapid dissociation of tissue samples, and thus less

degradation, than conventional methods using sharp blades for linear cutting of the

tissue.

In one aspect, the present invention provides a kit for tissue processing

comprising a flexible container with an access port for introducing solid particles

having a diameter sufficient to create a void space no smaller than the smallest cell to

be processed, a filter for separating processed cells and cell clusters from particles and

tissue debris, and a container or chamber to collect the processed cellular materials

and medium. Preferably the solid particles comprise beads selected from the group

consisting of glass, ceramic, natural minerals, plastic, or metal, and the dissociation

bag is transparent or translucent. Preferably, flexible container comprises a

transparent or translucent dissociation bag, and the access port is reversibly sealable.

In one embodiment, the dissociation bag is separated into two chambers by the

filter, with one chamber used to process the tissue sample and the other chamber to

collect the dissociated single cells and cell clusters. In an alternative embodiment of the kit, a collection bag is separated from the

dissociation bag by the filter, and the dissociation bag is used for processing the tissue

sample and the filter separates the dissociated cells and cell clusters, which are

collected in the collection bag, from the solid particles and tissue debris. In a further

embodiment, the kit further comprises a wash bag containing medium, inhibitors, or

enzymes, which is connected to the dissociation bag or to the collection bag to

provide wash solutions, enzymes, or enzyme inhibitors to the tissue sample, or to the

single cells or cell clusters.

In another aspect, the present invention provides a method for dissociating

solid tissues into viable single cells or cell clusters. The method comprises placing

the solid tissue together with an effective grinding amount of solid particles in a

tissue-compatible liquid in a flexible container which is in fluid connection with a

filter and contains an access port, and wherein the diameter of the solid particles is

large enough to create a void space no smaller than the smallest cell to be processed,

externally mechanically manipulating the solid particles in the flexible container to

grind the tissue into single cells or clusters of cells and produce a suspension of

dissociated tissue, and separating the single cells and clusters of cells from the solid

particles and tissue debris larger than the cells or cell clusters by passing the

suspension of dissociated tissue through the filter.

Preferably the solid particles comprise beads selected from the group

consisting of glass, ceramic, natural minerals, plastic, or metal.

In one embodiment of the method, the flexible container comprises a

dissociation bag comprising an access port for introducing tissue and tissue compatible medium into the dissociation bag and a filter for separating, via gravity

force, beads and debris larger than the cells or cell clusters from the cells or cell

clusters and subcellular debris. Preferably, the dissociation bag is transparent or

translucent and the access port is resealable. The dissociation bag is separated into two

chambers by the filter, with one chamber used to process the tissue sample and the

other chamber to collect the dissociated single cells and cell clusters.

In another embodiment, a collection bag is separated from the dissociation bag

by the filter, and the dissociation bag is used for processing the tissue sample and the

filter separates, via gravity force, the dissociated cells and cell clusters, which are

collected in the collection bag, from the solid particles and tissue debris. In an

additional embodiment, the method further comprises a wash bag containing medium,

inhibitors, or enzymes, which is connected to the dissociation bag or to the collection

bag to provide wash solutions or enzyme inhibitors to the tissue sample or single cells

or cell clusters.

Brief Description of the Drawings

The invention and several of its aspects may be better understood in relation to

the following Figures, wherein:

Figure 1 schematically shows the glass beads-tumor sample interface.

Figure 2 shows a prototype tumor processing kit.

Figure 3 shows a microscopic view of a histological section of processed primary

MCA-38 tumor within a Theracyte™ device. Figure 4 shows a microscopic view of a histological section of cultured MCA-38

cells within a Theracyte™ device.

Detailed Description Of The Preferred Embodiments

In one aspect of the present invention, a kit for preparing viable single cell

suspensions or clusters of cells from solid tissue samples while maintaining an aseptic

environment is disclosed. As used herein, the term "tissue samples" includes both

tumorigenic and nontumorigenic tissues. .As used herein, the term "closed

environment" refers to a container that is closed once the tissue sample is placed in

the system. The tissue processing kit comprises a flexible container for receiving

tissue in a tissue compatible medium, solid particles for grinding the tissue in the

flexible container. The flexible container comprises an access port for introducing the

tissue and the tissue compatible medium into the container and a filter for separating

particles and debris larger than the cells or cell clusters from cells, cell clusters and

subcellular debris.

In one embodiment of the method, the flexible container comprises a

dissociation bag that is transparent or translucent. Dissociation bag material properties

to be considered include, but are not limited to, clarity, ductility, flexibility, gas barrier

properties, mechanical strength, dimensional stability, residual leaching capability,

biocompatibility, usable temperature range, and seal processabilty, by either ultrasonics,

heat or radio frequency sealing. Suitable dissociation bag materials include hydrocarbon

plastics and elastomers, such as polyolefins (polyethylene, polypropylene), natural

rubber or synthetic elastomers; carbon-chain polymers, such as polystyrene, acrylic, polyvinyl esters (polyvinyl alcohol, polyvinyl acetyl, ethylene vinyl acetate copolymer);

heterochain thermoplastics, such as polyamides, polyester, polyether and cellulosic

polymers; and thermosetting resins, such as phenolic, amino, unsaturated polyester,

epoxy, polyurethane and silicone. In a preferred embodiment, the dissociation bag will

comprise a clear plastic (e.g. polyethylene) face for visual presentation and plastic

venting material like Tyvek® for aeration. This allows an operator to apply

customized, controlled compressive and shear forces during mechanical dissociation.

The operator is able to visually identify larger tumor sections, and localized particle

shearing action by manually manipulating the dissociation bag. Particle shearing

action is generated by the operator applying force to the dissociation bag, causing the

particles to be scrubbed against the surface of the tissue section.

In one embodiment of the kit, a collection bag, separated from the dissociation

bag by an in-line filter, is used for collecting the single cells or clusters of cells.

Alternatively, the dissociation bag is separated into two chambers by the filter, with

one chamber used to process the tissue sample and the other chamber to collect the

dissociated single cells and cell clusters. Filtration can be accomplished by methods

including, but not limited to, gravity force, vacuum, or pumps. In a preferred

embodiment, filtration is accomplished via gravity force.

In a further embodiment, the kit further comprises providing wash solutions,

enzymes or enzyme inhibitors to the tissue sample or the dissociated cells or cell

clusters, from a wash bag containing medium, inhibitors, or enzymes, which is in fluid

connection with the dissociation bag or with the collection bag. The collection and

wash bags comprise the same materials and properties as the dissociation bag. The solid particles of the kit preferably comprise glass, ceramic, natural

minerals, plastic, or metal beads. The solid particles are preferably greater in diameter

than the diameter of a single cell to be dissociated or single cell cluster, to permit

separation of the beads from the cells by a filter. The solid particles generally range in

diameter from between about 25 mm to about 400mm. In a preferred embodiment,

the beads range in diameter from between about 100 mm to about 250 mm. In a most

preferred embodiment, the beads are about 165 mm in diameter. Preferably, the ratio

of bead mass to cell mass is between 0.1 and 10. As the number of beads is

decreased, processing times will increase. The size of the beads can be varied, thus

varying the resulting void space between the beads.

A preferred embodiment of a tissue processing kit according to the present

invention is shown in Figure 2. The tissue mass is dissociated into cells and tumor

pieces in a dissociation bag 8. Single cells and'or small clusters are passed through a

separating filter 10 and tubing 12 via gravity force and are collected in a collection

bag 14, the contents of which can be centrifuged if a cell pellet is required.

Alternatively, filtration can be accomplished by other means, such as vacuum or pump

forces.

A wash bag 16 is optional and may contain wash solutions, enzymes or

inhibitors of any enzymes that may have been used in the process. Suitable wash

solutions include, without limitation, cell specific growth media, phosphate buffered

saline (PBS), Plasmalyte® and non-buffered saline. Suitable enzyme inhibitors depend

on the enzymes used for dissociation, and include, but are not limited to serum and specific enzyme inhibitors such as leupeptin and antipain. Such solutions, enzymes or

enzyme inhibitors are added to the dissociation bag 8 via tubing 18. Alternatively, the

wash bag 16 could be similarly connected to the collection bag 14 to provide wash

solutions or enzyme inhibitors to the single cells or cell clusters.

Preferably, access to the dissociation bag for reversible sealing is

accomplished using a threaded portal and cap designed with a mechanical seal to

isolate the bag contents.

The exact size and volume of the dissociation bag to be used will vary with the

size of the tissue sample to be processed. The dissociation bag must be of a size

appropriate for the mass of the tissue to be dissociated. The tissue must fit through

the portal of the dissociation bag and must fit within the bag. For smaller tissue

samples, the bag must be proportionately smaller so that the sample is not lost in the

void spaces.

Similarly, the separating filter 10 used will vary depending on the size of the

dissociated cells and cell clusters and the size of the solid particles used. The

separating filter 10 must allow the dissociated cells or cell clusters to pass from the

dissociation bag 8 into the collection bag 14, but must not allow the solid particles to

pass into the collection bag 14. The size of the collection bag will vary depending on

the size of the tissue mass to be dissociated. For example, if the tissue mass is very

small, then a small collection bag should be used in order to minimize tissue loss

during processing. In another aspect, the present invention teaches a method for producing single

cells or clusters of cells from solid tissue comprising placement of the solid tissue into

a flexible container containing an effective grinding amount of solid particles and a

tissue compatible liquid. The solid particles in the container are then externally

mechanically manipulated to grind the tissue into single cells or clusters of cells and

the single cells or clusters of cells are subsequently separated and collected. As used

herein, an effective grinding amount of solid particles means that number, type and

size of particles which grind the tissue into single cell and cell clusters upon

mechanical manipulation in less than sixty minutes, preferably less than 10 minutes.

The method allows greater surface areas of tissue to be dissociated at any one time

due to the increased particle/tissue contact surface area, as compared to traditional

linear cutting with a blade. Furthermore, tissue processing is conducted in a closed

environment, which may be maintained indefinitely and also allows both mechanical

dissociation and enzymatic digestion to occur simultaneously. As used herein, the

term "closed environment" refers to a container that is closed once the tumor is placed

in the system. The use of a closed environment further allows for tissue dissociation

to proceed in an aseptic environment.

The tissue compatible liquid may comprise any suitable liquid which maintains

viability of the cells sought to be dissociated from the tissue sample. The cell type or

properties of the tissue determines the type of media used. Suitable media include,

without limitation, cell specific growth media, phosphate-buffered saline ('TBS"),

Plasmalyte® and non-buffered saline. The method of the present invention utilizes mechanical dissociation by solid

particles. As shown in Figure 1 , solid particles 2 within a flexible container 4 mimic

the enzymatic digestion process by mechanically shearing the tissue sample only at

the surface of the sample 6. Traditional blade cutting methods are limited to linear

contact along the surface of a tumor section. The method of the present invention

allows greater surface areas of tissue to be dissociated at any one time due to the

increased particle/tissue contact surface area. Due to the increased number of contact

points, particle digestion may produce more rapid and uniform breakdown of tissue

versus traditional linear cutting action by sharp blades. Additionally, once tissue is

dissociated to a specific size it is less affected by the particle shearing action due to

gaps in the void space between particles. Therefore, this mechanical dissociation

process is less likely to damage the cells from over processing (i.e., degradation) as

long as the void space between particles is no smaller than the size of single cells.

In a preferred embodiment of the present invention, the solid particles

comprise glass, ceramic, natural minerals, plastic, or metal beads having a diameter

sufficient to create a void space between particles no smaller than the smallest cell to

be processed. The size of the beads can be varied, thus varying the resulting void

space between the beads. The beads are preferably greater in diameter than the single

cells or single cell clusters to be dissociated. The solid particles generally range in

diameter from between about 25 mm to about 400mm. In a preferred embodiment,

preferred embodiment, the beads are about 165 mm in diameter. The ratio of beads to cell mass should be between 0.1 and 10. As the number of beads used is decreased,

processing times will increase.

The flexible container preferably comprises a dissociation bag comprising a

reversibly sealable access port for introducing tissue, beads and tissue compatible liquid

in various ways, including but not limited to radiofrequency welding, ultrasonics, or

heat, depending on the material composition of the bag. In a preferred embodiment

access to the dissociation bag for sealing and resealing is accomplished using a threaded

portal and cap designed with a mechanical seal to isolate the bag contents.

In one embodiment, the dissociation bag is separated into two chambers by the

collect the dissociated single cells and cell clusters.

In an alternative embodiment, a collection bag is separated from the

dissociation bag by the filter. The dissociation bag is used for processing the tissue

collected in the collection bag, from the solid particles and tissue debris, which are

retained in the dissociation bag. In either embodiment, after manual dissociation of

the tissue sample, the suspension of dissociated tissue is drained by gravity through

the separating filter.

Ln a further embodiment, the method further comprises providing wash

solutions or enzyme inhibitors to the tissue sample or the dissociated cells or cell

connection with the dissociation bag or the collection bag. The present invention may be better understood with reference to the

accompanying Examples that are intended for purposes of illustration only and should

not be construed to limit the scope of the invention, as defined in the claims appended

hereto.

Example 1: Tumor Processing Procedure:

The tumor processing procedure in this Example was used in the following

examples, except where so noted. The processing systems used in the following

Examples varied depending on the experiment, as described below (Refer to Figure

2).

All steps were performed with aseptic technique in a clean, filtered aseptic

environment such as a Class II biological safety cabinet or equivalent, at room

temperature or lower, in order to minimize the activity of degredative enzymes.

Tissue processing can be carried out at almost any temperature, limited only by the

viability and function of the cells.

1. Glass beads and resected rumor in media were added to the flexible

dissociation bag. Note: enzymes were added where indicated.

2. The opening to the dissociation bag was heat sealed with a hand held bar

sealer.

3. The bead/tumor mixture in the dissociation bag was manually agitated. This

resulted in a rapid breakdown of the primary tumor sample. Agitation continued for

approximately 2-4 minutes or until sample was visually dissociated. 4. After agitation, the dissociation bag was connected with an in-line filter set

connected to a collection bag.

5. The suspension of dissociated tumor was drained by gravity through the

separating filter and captured in a collection bag.

6. After the suspension had drained, the tubing was clamped shut and the

collection bag was removed by cutting the tubing upstream of the clamp.

7. Cell suspension was transferred into a 50ml conical vial and allowed to gravity

settle for approximately 5 minutes.

8. The cell suspension was evaluated for viability (trypan blue exclusion) by

microscopic inspection.

Example 2: Dissociation of MCA-38 primary tumor without filtration

Primary tumor was initiated by subcutaneous bolus injection of lxl 0⁶

syngeneic mouse carcinoma cells (MCA-38) into C57B6 mice (Harlan). The cells

were a generous gift from Dr. Agusto Ochoa at the National Cancer Institute. After

approximately 10 days, animals were euthanized in accordance with the requirements

of the Animal Welfare Act and the National Institutes of Health guidelines for the care

of laboratory animals, and the tumors were resected for experimental use. Tumor was

stored in Dulbecco's modified Eagle's Medium (Sigma) supplemented 20% Fetal

Bovine Serum (Irvine Scientific), 1% Penicillin (10,000 Units/ml) Streptomycin

(lOmg/ml) (Sigma) and 1%L-Glutamine (200 mM (Sigma) and transferred to the

tumor processing system. The tumor processing system contained the following components (refer to

Figure 2):

Component Description

8 PL732 Blood Bag, 1 L, code 4R21 10 (Baxter)

Glass Beads, 0.065 inch diameter (165 millimeter) (Kimble)

10 None used in experiment

14 Cell Wash Set, code ITX-1015 (Baxter)

16 PL732 Blood Bag, 1 L, code 4R2110 (Baxter)

One corner of the dissociation bag was cut open with scissors. Approximately

10 grams of resected tumor combined from several animals was added to the

dissociation bag together with approximately 100 grams of sterile glass beads. A

digestive enzyme mix consisting of 40ml 0.25% Trypsin (Sigma), 10ml (200

Units/ml) Collagenase (Sigma) and 2 ml Fetal Bovine Serum (Harlan) was added to

the dissociation bag. The cut opening in the dissociation bag was heat sealed with a

hand held sealer (Portable Type 2 Poly, Packaging Aids Corporation). The bag was

agitated for approximately four minutes to enzymatically digest and mechanically

dissociate the tumor.

One comer of the wash bag was cut open with scissors, 250 ml of media as

described above was added to the wash bag, which was then heat sealed with a bar

sealer. A sterile tube connector (Terumo) was used to connect the tubing of

component A and D. The contents of the wash bag was drained into the dissociation

bag. Using the Terumo sterile tube connector, the tubing of the collection bag and the dissociation bag were connected. Approximately 50 ml of the cell suspension was

drained into the collection bag, which was then centrifuged at 2000 revolutions per

minute for 10 minutes to concentrate the cell suspension. The resulting cell

suspension was tested by trypan blue exclusion for viability. The cells were 46%

viable. The initial viability of the tumor mass was unknown, but certainly was less

than 100% due to necrosis. Thus, digestive enzymes and glass beads in the

dissociation bag successfully enzymatically digested and mechanically dissociated the

tumor into a single cell suspension after approximately four minutes of agitation.

Example 3: Dissociation of MCA-38 primary tumor with filtration

MCA-38 primary tumor was initiated and harvested as described in Example

2.

The tumor processing system contained the following components (refer to Figure 2):

Component Description

8 PL732 Blood Bag, 1 L, code 4R21 10 (Baxter)

Glass Beads, 0.065 inch diameter (165 millimeter) (Kimble)

10 In-Line Filter, code 2C7600 (Baxter)

14 Cell Wash Set, code ITX- 1015 (Baxter)

16 PL732 Blood Bag, IL, code 4R2110 (Baxter)

Resected tumor was enzymatically digested and mechanically dissociated as

described in Example 2. An in-line filter was connected to the collection bag with a sterile tube connector (Terumo). The in-line filter was connected by sterile spike

adapter to the dissociation bag and approximately 50 ml of the cell suspension was

drained into the collection bag, which was then centrifuged at 2000 rpm for 10

minutes to concentrate the cell suspension. Digestive enzymes and glass beads in the

dissociation bag successfully dissociated the tumor into a single cell suspension and

the filter allowed for easy separation of the cell suspension from the remaining tissue

debris. The cells appeared intact by microscopic evaluation (not shown).

Example 4: Dissociation of primary human glioblastoma

Primary human glioblastoma was received from the University of Illinois at

Chicago (U1C). It was stored and transferred in saline. The tumor processing system

was the same as described in Example 3. Approximately 2.5 grams of resected tumor

was dissociated as described in Example 2.

The in-line filter was connected to the collection bag with a sterile tube

connector (Terumo®). The in-line filter was connected to the dissociation bag and

approximately 50ml of the cell suspension was drained into the collection bag.

Digestive enzymes and glass beads in the dissociation bag successfully dissociated the

tumor into a single cell suspension and the filter allowed for easy separation of the

cell suspension from the remaining tissue debris. The cells appeared intact by

microscopic evaluation (not shown).

Example 5: Dissociation of Bl 6 primary tumor

B16 melanoma tumor, derived from cells obtained from the American Type Tissue Collection (ATCC; Accession No. CRL6323) was initiated and harvested as

described in Example 2. The tumor processing system contained the following

components (refer to Figure 2):

Component Description

8 PL732 Blood Bag, IL, code 4R21 10 (Baxter)

Glass beads, 0.065 inch diameter (165 millimeter) (Kimble)

10 In-line Filter, code 2C7600 (Baxter)

14 Cell Wash Set, code ITX-1015 (Baxter)

16 None used in experiment

One corner of the dissociation bag was cut open with scissors. Approximately

10 grams of resected tumor from several animals was added to the dissociation bag

together with approximately 200 grams of glass beads and 40ml of media. Additional

beads were included because more tumor was processed. No digestive enzyme mix

was used. The cut opening in the dissociation bag was heat sealed with a hand held

sealer (Portable type 2 Poly, Packaging Aids Corporation). The bag was agitated to

dissociate the tumor.

The in-line filter was connected to the collection bag with a sterile tube

connector (Terumo). The in-line filter was connected to dissociation bag and

approximately 50ml of the cell suspension was drained into the collection bag. Glass

beads in the dissociation bag successfully dissociated (without enzyme) the tumor into

a single cell suspension and the filter allowed for easy separation of the cell suspension from the remaining tissue debris. The cells appeared intact by

microscopic evaluation (not shown).

Example 6: Dissociation of Bl 6 primary tumor

B16 melanoma tumor was initiated and harvested as described in Example 2.

Cells were dissociated with the same procedure as Example 5. The results were the

same, and thus the processing system reproducibly dissociates (without enzyme

dissociation) a B 16 primary tumor into a single cell suspension, with easy separation

of the cell suspension from the remaining tissue debris.

Example 7: Dissociation of RIP-Tag insulinoma tumor

Primary insulinoma tumor containing the rat insulin promoter under the

control of SV40 T antigen (RlP-tag; obtained from Jackson Labs) was initiated and

harvested from athymic mice as described in Example 2. The tumor processing

system contained the following components (refer to Figure 2):

Component Description

8 PL732 Blood Bag, 1 L, code 4R21 10 (Baxter)

Glass Beads, 0.065 inch diameter (165 millimeter) (Kimble)

10 In-line Filter, code 2C7600 (Baxter)

14 Cell Wash Set, code ITX(Baxter)

16 None used in experiment Resected tumor w as dissociated and collected as described in Example 5.

Glass beads in the dissociation bag successfully dissociated (without enzymes) the

insulinoma tumor into a single cell suspension and the filter allowed for easy

separation of the cell suspension from the remaining tissue debris. Cell viability was

assessed with trypan blue exclusion by microscopic evaluation. Cell viability was

approximately 63%. Samples were submitted to histology prior to and after

processing for staining. Staining for presence of insulin and SV-40 T-antigen were

positive in all samples. This implies the cells functioned prior to and after undergoing

the tumor processing system.

Example 8: Dissociation of MCA-38 primary tumor using a different dissociation

bag

MCA-38 primary tumor was initiated and harvested as described in Example

2. The tumor processing system contained the following components (refer to Figure

2):

Component Description

8 All-In-One Container, 500ml, code 2B8152 (Baxter-Clintec) Glass

Beads, 0.065 inch diameter (165 millimeter) (Kimble)

10 In-line Filter, code 2C7600 (Baxter-INS)

14 Cell Wash Set, code ITX-1015 (Baxter-Fenwal)

16 None used in experiment MCA-38 cells were dissociated and collected with the same procedure as

Example 5, and the results were the same. Thus, the process was reproducible using a

different dissociation bag than was used for Examples 2 and 3.

Example 9: Dissociation of RIP-Tag insulinoma primary tumor

RIP-Tag insulinoma primary tumor was resected and dissociated with the

same procedure as Example 7. The tumor processing system used was the same as

described in Example 8, and the results were the same. Thus, the processing system

reproducibly dissociates the insulinoma rumor into a single cell suspension, with easy

separation of the cell suspension from the remaining tissue debris.

Example 10: Dissociation of B16 primary tumor

B 16 melanoma tumor was initiated and harvested as described in Example 2.

Component Description

8 All-In-One Container, 500ml, code 2B8152 (Baxter-Clintec) Glass

Beads, 0.065 inch diameter (165 millimeter) (Kimble), or Stainless

Steel beads, 0.065 inch diameter (165 millimeter) (Hartford Ball)

10 In-line Filter, code 2C7600 (Baxter-IVS)

14 Cell Wash Set, code ITX-1015 (Baxter-Fenwal)

16 None used in experiment B 16 cells were dissociated and collected with the same procedure as in

Example 5. Samples were dissociated with glass beads or stainless steel beads to

compare the components. The results were the same, the tumor cell suspension

looked intact by microscopic evaluation. Thus, the process is reproducible with

different types of dissociation beads

Example 11: Tumorigenic potential of processed B 16 primary tumor cells

This experiment was conducted to determine if processed B16 primary tumor

remained tumorigenic potential (i.e., whether they remained functional). B16 primary

tumor processed in Example 5 was cultured in 150 cm² flasks in culture media (as

described in Example 2) for approximately one month at 5% CO, and 37° C. Cells

were trypsinized and pooled in a 50 ml conical tube. Cells were counted and

resuspended at 5xl0⁵ cells per 50 μl of media. 50 μl of cell suspension was injected

into the dorsal subcutaneous space of ten C57B6 mice (Harlan). Animals were

inspected periodically for the appearance of tumor at the injection site. After thirteen

days, all ten mice developed observable tumor. Thus, cell function, as measured by

tumorigenicity of processed B16 cells, was retained after processing.

Example 12: Survival and Proliferation of processed MCA-38 primary tumor cells This experiment was conducted to determine if processed MCA-38 primary

tumor was able to survive and proliferate in a implant assembly such as Baxter

Healthcare Corporation's immunoisolation Theracyte™ device, described in Geller et al, Journal of Immuno therapy 20:131-137 (1997), herein incorporated by reference

Primary tumor was initiated by subcutaneous bolus injection of lxl 0⁶ syngeneic

mouse carcinoma cells (MCA-38) into C57B6 mice (Harlan). After approximately 10

days, animals were anesthetized and tumors were resected for experimental use.

Tumor was stored in Dulbecco's modified Eagle's Medium (Sigma) supplemented

20%) Fetal Bovine Serum (Irvine Scientific), 1% Penicillin (10000

Units/miyStreptomycin (10 mg/ml)(Sigma) and 1% L-Glutamine (200 mM)(Sigma)

and transferred to the tumor processing system

Component Description

A All-In-One Container, 500ml, code 2B8152 (Baxter-

Clintec) Glass Beads, 0.065 inch diameter (165

millimeter) (Kimble)

B In-line Filter, code 2C7600 (Baxter-INS)

C Cell Wash Set, code ITX-1015 (Baxter-Fenwal)

D None used in experiment

MCA-38 cells were dissociated and collected with the same procedure as Example 5. Briefly, one corner of component A was cut open with scissors. Resected

tumor from several animals was added to the dissociation bag. Approximately 200

grams of glass beads and 40 ml of media were added to the dissociation bag. No digestive enzyme mix was used. The cut opening in the dissociation bag was heat

sealed with a hand held sealer (Portable Type 2 Poly, packaging Aids Corporation).

The bag was agitated to dissociate the tumor.

Component B was connected to component C with a sterile tube connector

(Terumo). Component B (in-line filter) was connected to component A and

approximately 50ml of the cell suspension was drained into component C. Glass

a single cell suspension and the filter allowed for easy separation of the cell

suspension from the remaining tissue debris. Cell viability was assessed with trypan

blue exclusion by microscopic evaluation. 2.35x10⁸ viable cells were present in the

resulting suspension. Cells were resuspended to a final concentration of lxlO⁷

cells/20 μl of media. As a control, cultured cells were harvested and suspended in

saline at a concentration of lxlO⁷ cells/20 μl of saline.

20 μl of cell suspension were loaded into a Theracyte™ device by injection

into the port of the device using a 25 μl Hamilton syringe with a blunt needle. The

devices were then sealed using a silicone adhesive (Dow Corning, Midland, MD)

injected into the port. The loaded devices were placed in a 150 x 15 mm petri dish

until implantation. 16 devices were loaded with primary cells and 16 devices were

loaded with cultured MCA-38 cells. Devices were implanted on the dorsal surface in

the subcutaneous space in C57B6 mice (each mouse received two devices containing

the same cell type). Ten days post implantation, ten devices containing cultured cells

and four devices containing processed primary tumor cells were explanted, histologically processed and stained with hemotoxylin and eosin. Histological cross

sections of the devices were microscopically evaluated for cell viability within the

lumen. Devices loaded with either cultured or primary processed tumor cells

contained approximately six to eight layers of viable cells within the lumen. Fourteen

days post implantation, two devices containing cultured cells and four devices

containing processed primary tumor cells were explanted and histologically

processed. These devices displayed similar histological profiles to the ten day sample

set. The remaining devices were not explanted due to animal death. The histology of

TheraCyte™ devices explanted at fourteen days is shown in Figures 3 and 4.

Processed MCA-38 primary tumor looked approximately the same as cultured MCA-

38 cells in this experiment. These results demonstrate that processed MCA-38

primary tumor survived and proliferated in a TheraCyte™ device.

Claims

We claim:

1. A kit for processing tissue and separating single cells or cell clusters from

tissue debris comprising:

(a) a dissociation bag for receiving tissue in a tissue compatible medium,

wherein the flexible bag comprises an opening for introducing tissue and tissue

compatible medium into the flexible bag;

(b) solid particles in the dissociation bag having a diameter sufficient to

create a void space no smaller than the smallest cell to be processed, for grinding the

tissue in the dissociation bag; and

(c) a filter for separating the solid particles and debris larger than the cells

or cell clusters from the cells or cell clusters and subcellular debris; and

2. The kit of claim 1, wherein the dissociation bag is transparent or translucent.

3. The kit of claim 1 further comprising a collection bag, separated from the

flexible bag by the filter.

4. The kit of claim 1, wherein the solid particles comprise beads selected from

the group consisting of glass, ceramic, natural minerals, plastic, or metal.

5. The kit of claim 1, wherein the flexible bag is separated into two chambers by

the filter.

6. The kit of claim 1, wherein the opening in the flexible bag is reversibly

sealable.

7. The kit of claim 1, further comprising a wash bag which is in fluid connection

with the dissociation bag, wherein the wash bag provides wash solutions or enzyme

inhibitors to the tissue sample.

8. The kit of claim 3, further comprising a wash bag which is in fluid connection

with the collection bag, wherein the wash bag provides wash solutions or enzyme

inhibitors to the dissociated cells or cell clusters.

9. A method for processing tissue and separating single cells or cell clusters from

tissue debris comprising:

(a) placing the solid tissue together with an effective grinding amount of

solid particles in a tissue-compatible liquid in a dissociation bag, wherein the

dissociation bag is in fluid connection with a filter and contains an access port, and

wherein the diameter of the solid particles is large enough to create a void space no

smaller than the smallest cell to be processed;

(b) externally mechanically manipulating the solid particles in the

dissociation bag to grind the tissue into single cells or clusters of cells and produce a

suspension of dissociated tissue; and

(c) separating the single cells and clusters of cells from the solid particles

and tissue debris larger than the cells or cell clusters by passing the suspension of

dissociated tissue through the filter.

10. The method of claim 9, wherein the solid particles comprise beads selected

from the group consisting of glass, ceramic, natural minerals, plastic, or metal.

11. The method of claim 9, wherem the flexible bag is transparent or translucent.

12. The method of claim 9 further comprising a collection bag which is separated

from the dissociation bag by the filter, for the collection of the single cells and clusters

of cells.

13. The method of claim 9, wherein the dissociation bag is separated into two

chambers by the filter.

14. The method of claim 9, wherein the access port in the dissociation bag is

reversibly sealable.

15. The method of claim 9, further comprising providing wash solutions or

enzyme inhibitors to the tissue sample in the dissociation bag from a wash bag which

is in fluid connection with the dissociation bag.

16. The method of claim 12, further comprising providing warh solutions or

enzyme inhibitors to the dissociated cells or cell clusters in the collection bag from a

wash bag which is in fluid connection with the collection bag.