WO2022094613A1

WO2022094613A1 - Cell isolation device and methods of use

Info

Publication number: WO2022094613A1
Application number: PCT/US2021/072138
Authority: WO
Inventors: Roger ESTEBAN VIVES; Peter ZINNBAUER; Arne Schulz; Robin Augustus ROBINSON; Frank Schubert
Original assignee: Renovacare Sciences Corp.
Priority date: 2020-10-29
Filing date: 2021-10-29
Publication date: 2022-05-05

Abstract

Small fractions of cells obtained from tissues and organs have a high potential to regenerate whole organs or tissues or parts of organs and tissues without altering the function of the primary source. Autologous cell transplantation has several advantages in comparison with allogeneic cell transplantation as the low immune-rejection, and fewer cell processing steps, and no storage process. Disclosed herein is a cell isolation device, systems and methods that use mechanical and enzymatic steps to isolate cells from an autologous biopsy tissue that is minimally processed. The device comprises a tissue digestion unit, a cell sieve unit and a filter membrane. The cell isolation device requires minimal human manipulation to isolate the cells from a small biopsy, while the automatic steps under a sterile environment allow a maximum cell harvesting and avoiding/minimizing cell damage. Isolated cells can be used to treat diseases, disorders, and conditions, particularly diseases, disorders, and conditions associated with the type of tissue from which the cell was derived.

Description

CELL ISOLATION DEVICE, SYSTEM, AND METHODS OF USE

CLAIM FOR PRIORITY

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial Number 63/107,387, filed on October 29, 2020, and U.S. Provisional Patent Application Serial Number 63/127,744, filed on December 18, 2020, the benefit of priority of which are claimed hereby, and which are incorporated by reference herein in their entirety.

TECHNICAL FIELD

[0002] This disclosure relates to systems, devices, and methods for isolating cells from tissues.

BACKGROUND

[0003] Autologous cell-spray grafting of non-cultured epidermal-dermal cells is an innovative stem cell-based therapy [1] that uses a small skin donor site biopsy and thus presents an early treatment alternative to conservative treatment for large deep partial -thickness burns [2], By employing epidermal and dermal progenitor cells and using the patient’s wound as a “bioreactor” for cell expansion in situ, re-epithelialization can occur with a relatively small number of cells (as compared to split-thickness skin grafts (STSG) or cultured epidermal autografting) [3],

[0004] In the case of skin transplantation, the technique has evolved from autologous full-thickness grafts to STSG, to mesh expansion of STSGs into a “skin lattice” [4-6], The most widely used technique limits the typical expansion of a STSG donor area to 1 :3, as larger ratios are often associated with unsatisfactory results and complications [7-9], The Meek technique [10] cuts the STSG into small tissue cubes, enabling a donor site to wound ratio from 1 :3 to 1 :9 [11], For large burn wounds, however, the lack of available donor site remains a problem.

[0005] Manual cell isolation using a two-step enzyme process was described herein in the literature for skin derived cell expansion [12], Moreover, the cell isolation and in vitro cell expansion technique was used clinically to treat burned patients [3, 13], The same manual cell isolation was subsequently used to clinically treat patients without the need of an in vitro expansion step [14], The further addition of an extra enzymatic digestion step recovered the dermal fraction [2, 15],

[0006] This method provides an on-site cell isolation process followed by the immediate application of autologous stem and progenitor cells to a freshly debrided wound bed. Cells in a saline suspension are sprayed uniformly across the wound bed to proliferate and accelerate the re-epithelialization process [2, 3, 14], However, the current cell isolation process is totally manual depending on the personnel and being subject to human error or trained personnel availability. [0007] There is a long felt need in the art for cell isolation devices useful for efficiently isolating cells from tissue or organs for use in vivo or in vitro, particularly cells with regenerative capacity. Cell isolation can require multiple units and numerous processing steps. With opening and closing units multiple times and/or transferring solutions/materials between units or tubes, the process can create contamination risks. There is a need for a cell isolation process with reduced steps and a cell isolation device with a simplified design. The present invention satisfies these needs.

SUMMARY

[0008] The present inventors recognize, among other things, an opportunity for providing a quick and easy device and method to isolate cells from tissues with minimal human input.

[0009] It is known that small fractions of cells obtained from tissues and organs have a high potential to regenerate whole organs or parts of organs without altering the function of the primary source. Autologous cell transplantation has several advantages over allogeneic cell transplantation, such as low immune-rejection, fewer cell processing steps, and no storage process. [0010] The present application discloses a device useful for isolating cells from tissues and organs and providing viable cells as a single cell suspension, where a single cell suspension would be a suspension of cells that includes primarily single cells or a few small clusters/conglom erates of cells and includes relatively few or no large clusters/conglomerates of cells. The isolated cells can then be used for treatment of a subject in need thereof or they can be used ex vivo or in vitro. In one aspect, the isolated cells can be sprayed using a cell spray gun as described in U.S. Pat. No. 9,505,000, U.S. Pat. No. 9,610,430, U.S. Pat. No. 10,376,658, U.S. Pat. No. 11,040,363, PCT Patent Application No. PCT/US2017/037274, PCT Patent Application No. PCT/EP2017064094, and German Patent No. DE102011100450B4.

[0011] In one aspect, the cell isolation device uses mechanical and enzymatic steps to isolate cells from biopsy tissue. In one aspect, the biopsy tissue undergoes minimal processing relative to other techniques. In one aspect, the tissue is autologous. In another aspect, the tissue is allogeneic. The cell isolation device requires the least possible human manipulation to isolate the cells from a small biopsy, while the automatic steps under a sterile environment allow maximum cell harvesting and low cell damage.

[0012] The present application provides a cell isolation device which comprises, in one aspect, a tissue digestion chamber, a biopsy holder, and a cell filter/sieve unit. In one aspect, the cell isolation process uses sterile enzymes and solutions. The process allows for washing the tissue and cells without a centrifugation step.

[0013] In one embodiment, a final wash or suspension solution can be used that has additional ingredients added or the cells can be applied to a material or solution to be administered to a subject wherein that material or solution may contain additional ingredients or materials. The additional ingredients can include, but are not limited to, biologically active molecules, antibodies, growth factors, cytokines, hormones, drugs, antibiotics, gels, hydrogels, matrix materials and matrices, including extracellular proteins, carriers, wound dressings, scaffolding, and combinations thereof, as well as agonists and antagonists of each of these agents if available and useful. Some of the above, such as certain wound dressings and scaffolding can serve as the delivery agent and the cells can be applied to them as the delivery vehicle.

[0014] One embodiment is illustrated in FIG. 1.1a. The FIG. discloses an exemplary cell isolation device, including its subunits - a tissue digestion chamber, a biopsy holder, and a cell sieve (or filter) unit. In one aspect, the cell isolation device can be subdivided into multiple parts or chambers. Illustrated in 1.1a is one embodiment consisting of three mam components- a cell isolation chamber (or tissue digestion chamber), a biopsy holder, and a cell sieve unit (or filter unit). Optionally, there is no biopsy holder. Multiple inlets and outlets can be provided in the cell isolation device and can be used for input of washing solutions, waste/output of wash solutions, and an opening to a cell sieve filtration unit where cells are extracted from the digestion core. The cell sieve unit demonstrated has three separate filters and the membranes can be of differing pore sizes. Optionally, the number of filters can be varied.

[0015] In one embodiment (see FIG. 1. lb), a cell isolation device can comprise a chamber connecting the filter/sieve unit on the left with the tissue digestion chamber and biopsy holder on the right. In another embodiment, (see FIG. 1.1c) an exemplary cell isolation device can comprise a mechanism to open or close a channel between the filter/sieve unit on the left with the rest of the unit on the right.

[0016] In one aspect, the cell isolation device comprises a biopsy holder (see, for example, FIGS. 1.1 to 4). In one aspect, the biopsy holder is removable. In one aspect, there is not a biopsy holder and the biopsy tissue can be placed directly into the tissue digestion chamber (rather than placing the biopsy tissue in the biopsy holder and then placing the biopsy holder in the tissue digestion chamber).

[0017] In one embodiment, the cell isolation device is a cylindrical unit. In one embodiment it is box or cuboid shaped. In another embodiment it is flat. In one aspect, a cell isolation/tissue digestion chamber has three ports for air liquid interchange: an inlet port that allows pumping enzymes (1) and saline solution, an air vent valve to keep the correct inner pressure (2), and an outlet port to evacuate waste products (3). The capacity of the cell isolation/tissue digestion chamber can vary, including, but not limited to, volumes such as about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 300 mL, 500 ML, 1 L, or more. During the cell digestion process the inlet port allows the tissue digestion chamber filling by pumping an appropriate isotonic solution or buffered salt solution containing an enzyme of choice such as an enzyme/Lactated Ringer’s Solution (LRS) while the air valve port keep the inner pressure constant. The waste outlet port allows emptying of the tissue digestion chamber by pumping out the used enzymes or the LRS cell washes. In the mam body of the tissue digestion chamber fits the biopsy holder which in turn keeps the tissue biopsy secure during for the tissue digestion process. A port is indicated on the upper part of the schematic and labelled on the schematic is an embodiment where the filter is integrated into the chamber. Optionally, more than one biopsy cassette (holder) can be used in the chamber.

[0018] In one embodiment (see FIG. 1.3a-1.3e), the cell isolation device comprises one or more waste outlets and can comprise various filtering membranes, including one or more filters. In one aspect, a waste outlet of the tissue digestion chamber has about a 5.0 pm pore-size membrane (also referred to herein as a waste filter membrane) to allow fluid interchange without losing the isolated cells (see FIGS. 1.3a, b, c, and d). The pore size of the waste filter membrane can range from about 0.4 to about 5.0 pm. In one aspect, the one or more filters can have a lid or a cap. Various methods and designs are provided to ensure fluid tight sealing to avoid dead spaces outside the filters. In one aspect, two or more filters are joined together. In one aspect, the digestion chamber and filter housing are connected as one single housing. In one aspect, a separate membrane filter is provided relative to the grid to provide a waste outlet to allow more filter surface and to reduce clogging of grid comers with debris. In one aspect, one function of the cell isolation device is to provide housing to the biopsy holder, enzyme interchange, cell wash steps with saline solution, and to maintain the cells under an isotonic solution until they are drawn into a second device (e.g., a syringe, cell collection unit or device, cell maintenance unit, applicator such a cell sprayer, or the like).

[0019] In one aspect, a biopsy holder can function to hold in place a skin biopsy inside the tissue digestion chamber during its enzymatic digestion process, mechanical tissue separation, and wash steps (see FIGS. 2a and 2b). In one aspect, the biopsy holder can be opened to insert the skin biopsy, wherein the biopsy has been placed against or rolled over the inner core/cylinder. In one aspect, the inner cylinder/core is removable, and a biopsy can be applied to it and then the inner cylinder can be inserted into the biopsy holder. The biopsy holder can then be closed (FIG. 2b). FIGS. 2a, 2b and 4a demonstrate the biopsy holder and its core unit. In one aspect, the biopsy holder and its core can be cuboid in shape.

[0020] In one aspect, the biopsy holder can be a metal rod having one or more clips attached thereto for securing the skin biopsy. In one aspect, the tissue digestion chamber is free of a biopsy holder and the skin biopsy can be free floating inside the chamber.

[0021] In one aspect, the tissue digestion chamber is designed to contain two or more biopsy holders. The two or more biopsy holders can be placed end to end (series) or in parallel.

[0022] In one embodiment, the tissue digestion chamber can include one or more anti-foam structures to break up foam or bubbles formed in the tissue digestion chamber when air is injected into the tissue digestion chamber. The air can be injected into the chamber to agitate or disrupt the tissue of the skin biopsy and promote digestion.

[0023] In one embodiment, a cell sieve unit is a cylindrical device that contains one or more sieves (or filters). In one aspect, there are two sieves/filters. In another aspect, there are three sieves/filters. In one aspect, when there is more than one sieve, the pore sizes of the sieve membrane decrease in size from one membrane to the next. For example, in one aspect, with three sieve membranes the pore sizes can be 200, 100 and 70 pm. This allows for sequentially passing cells through filters while excluding from the final cell solution tissue fibers, debris, and large clusters (aggregates) of cells that might inhibit their flow during disbursement into the second device as well as clog a cell sprayer device being used to administer the isolated cells.

[0024] A filter (sieve) housing unit is provided to contain membranes and can house one or more membranes (filters). The filter housing unit receives outflow of digested tissue from the cell digestion chamber and can be connected to that chamber in several ways (see FIGS. 4a-4d).

[0025] The filter housing for the filter unit can be made of any suitable materials. The membrane can be of any suitable material, such as those made by Millipore or PALL. Useful materials for filtration membranes can include, but are not limited to, nylon, polyethersulfone (PES), cellulose acetate (CA), cellulose nitrate (CN), regenerated cellulose (RC), polytetrafluoroethylene (PTFE), glass fiber, and acrylic copolymer. In one aspect, the membranes of the filters, when multiple filters are used in series, have decreasing pore size with the first being useful for excluding larger tissue debris and large cell aggregates and the next filter(s) in the series having a smaller pore size than the previous filter. Pore size of the filters/membranes can be from about 5.0 microns to about 200 microns, including 20, 25, 50, 75, 100, and 150. In one embodiment, the cell isolation device comprises three or more filters. In another it comprises two or more filters, and in another only one filter. The filters are useful for eliminating larger debris when using larger pore sizes and by reducing pore size of subsequent filters aggregates (clusters) of cells can be filtered out to produce a solution comprising substantially single cells and not (large) aggregates of cells. [0026] In one embodiment, some components of the system can be temperature regulated and can include maintaining a temperature of about 4°C, about 37°C, or a range of about 4°C to about 37°C, depending on whether the cell, tissue, or solution needs to be chilled or heated.

[0027] It is recognized that in other examples, each of the device components can have more or less connecting elements. It is recognized that the devices/units/components described do not have to be coupled together using the specific combination of features shown in the FIGS, and that other elements or designs can be used in addition to or as an alternative to the features shown in the FIGS.

[0028] In one embodiment, one or more of the components of the device can be vacuum casted. In addition, or as an alternative to vacuum casting, three- dimensional (3D) printing can be used for forming all or parts of the device.

[0029] Useful materials for 3D printing include, but are not limited to, MED610, MED620, PC-ISO (Polycarbonate), PA2200 (Polyamide), PA2201 (Polyamide), ABS-M30i (Acrylonitrile-butadiene-styrene), ABS-M30 (Acrylonitrile-butadiene-styrene), PA 650 (Polyamide), PA 615 GS (Polyamide), Accura Xtreme, Somos Watershed XC1122, Elastic Resin, MED625FLX, MED670, and MED690.

[0030] Useful materials for vacuum casting include, but are not limited to, SG 95 Shore D82 (Polyurethane), 7140 Shore A40 (Polyurethane), 7150 Shore A50 (Polyurethane), 7160 Shore A60 (Polyurethane), 7170 Shore A70 (Polyurethane), 7180 Shore A80 (Polyurethane), 7190 Shore A90 (Polyurethane), 8020 Shore A50-60 (Polyurethane), G 48 Shore D80 (Polyurethane), Biresin U1419 Shore A98 (Polyurethane), Biresin VG 230 Shore D82 (Polyurethane), Biresin VG 240 Shore D83 (Polyurethane), HPE Shore A40-D55 (Polyurethane), PR 777 Shore D75 (Polyurethane), PR 700 Shore D80 (Polyurethane), PRC 1810 Shore D85 (Polyurethane), PX 205 Shore D70 (Polyurethane), PX 212 Shore D76 (Polyurethane), PX 215 Shore D74 (Polyurethane), PX 331 Shore D87 (Polyurethane), PX 245 Shore D85 (Polyurethane), PU8150 (Polyurethane), UP5690 (Polyurethane), PU8434/8400 (Polyurethane), PX527/521 (Polyurethane), PU8743 (Polyurethane), PU8260-FR (Polyurethane), and PU8098 (Polyurethane).

[0031] Useful compounds for injection molding include, but are not limited to, ABS (Acrylonitrile-butadiene-styrene), PC (Polycarbonate), PC-ABS (Polycarbonate - Acrylonitrile-butadiene-styrene), PMMA (Polymethyl methacrylate), POM (Polyoxymethylene), PA (Polyamide), PA6 (Polyamide 6), PA6 + GF (Polyamide 6 - glass fiber reinforced), PA66 (Polyamide 66), PP (Polypropylene), PPO (Polyphenylene oxide), PPS (Polyphenylene sulphide), PPSU (Polyphenyl sulfone), PEEK (Poly etheretherketone), HDPE / LDPE (High density polyethylene / low density polyethylene), PE (Polyethylene), PET (Poly- ethylene-terephthalate), PES (Polyethersulfone), PVC (Polyvinyl chloride), PTFE (Polytetrafluoroethylene), and MBS (Methacrylate-butadiene-styrene terpolymer).

[0032] The cell isolation device and system as described herein are useful for isolating cells from various types of tissues. In one aspect, the tissue is obtained via a biopsy. In one aspect the tissue is skin. In one aspect, the tissue biopsy can be from any other organ or tissue, including, but not limited to, liver, lung, kidney, skeletal muscle, cardiac muscle, vessels, ducts, bone, bone marrow, adipose tissue, intestinal tissue, and spleen. In one aspect, once the cells are isolated from the biopsy tissue, they can then be administered to a subject in need thereof to replenish or replace diseased or lost cells of the parent tissue. When possible, the biopsy tissue or cells obtained from the subject are normal and following isolation using the systems and methods of the invention are then used to replace their diseased or dead counterpart cells. That is, in one aspect cells isolated using the systems, devices, and methods of the invention can be used to treat diseases, disorders, and conditions, particularly diseases, disorders, and conditions associated with the type of tissue from which the cell was derived.

[0033] In an example, the cells isolated using the cell isolation devices described herein can range from about 5 microns to about 70 microns. Although large aggregates of cells can be filtered out in the cell sieve unit, small cell aggregates of 70 microns or less (depending on the size of the last sieve) can be collected with the single cells, if they are not separated into a single cell suspension during the digestion or washes described below.

[0034] Isolated cells of the invention can be used with wound dressings to treat wounds. In one aspect, the wound dressing is passive and in another it is active. While passive wound dressings simply serve a protective function, active dressings promote healing through the creation of a moist wound environment. Interactive wound dressings, on the other hand, not only create a moist wound environment but also interact with the wound bed components to further enhance wound healing. For example, interactive wound dressings may reduce colonization count, reduce the level of exudate, improve wound bed moisture retention, improve wound collagen matrix, remove cellular products or provide protection for the epithelializing bed. Active wound dressings may use a mesh of interwoven capillaries to deliver a flow of fluids (wound perfusion), growth factors, nutrients, regenerative molecules, antibiotics, and other wound-healing agents. Wound dressings have multiple applications, including, but not limited to, diabetic foot ulcers, pressure ulcers, lower limb ulcers, and difficult to heal wounds.

[0035] Sieve and filter are used interchangeably herein. The term “mesh” is also used in the art, particularly for more coarse filtering.

[0036] Depending on the type of tissue being used, one or more enzymes can be used to isolate cells of interest from the tissue. Useful enzymes include, but are not limited to, gentlyase, dispase, collagenase (including Types 1, 2, 3, and 4), thermolysin, trypsin, trypsin-EDTA, elastase, hyaluronidase, papain, pronase, pancreatin, chymotrypsin, deoxyribonuclease I, and trypsin inhibitor, or a combination thereof. [0037] Enzyme inhibitors can also be used during the process and various inhibitors are available for use depending on the enzyme being used.

[0038] Useful physiologically acceptable solutions include, for example, Ringer’s lactate and phosphate buffered saline.

[0039] In an example, a spray deposition system can be used for delivery of skin cells suspended in a solution. The cells in the suspension can be single cells, small cell aggregates, or a combination of single cells and small cell aggregates. For purposes herein, a single cell suspension is a suspension would be a suspension of cells that includes primarily single cells or a few small clusters/conglomerates of cells and includes relatively few or no large clusters/conglomerates of cells. However, it is recognized that, because of the cell preparation procedures, cells can occasionally remain stuck together and form cell aggregates. One of ordinary skill in the art can modify the procedures to ensure that the desired proportion of single cells is obtained and used in a treatment solution.

[0040] In an example, at least two cell sources suitable for use in resurfacing and regeneration of damaged tissue can be used with the spray deposition systems described herein: (i) non-autologous cells, including stem and progenitor cells, and (ii) autologous cells, including the patient's own stem and progenitor cells. The cells can be suspended in solution to form the treatment solution - once formed, the solution can be transported to and contained in the second device for use in the spray deposition systems described herein. In some aspects, the second device can be a container or administration delivery vehicle such as a syringe, an eye dropper type of device or a pipette.

[0041] In an example, a method for preparing an autologous cell suspension can include harvesting tissue from a patient by means known in the art of tissue grafting, which can include taking a tissue biopsy. With the harvesting of the biopsy, consideration can be given to the depth of the biopsy and size of the surface area. The depth and size of the biopsy can influence the ease at which the procedure can be undertaken and the speed with which a patient can recover from the procedure. The chosen donor site may appropriately match the recipient site, for example post-auricular for head and neck, thigh for lower limbs, inner-upper-arm for upper limbs, or palm for sole or vice-versa. [0042] Once a biopsy has been harvested from a subject, the tissue sample can be subjected to physical and/or chemical dissociating means capable of dissociating cellular stratum in the tissue sample. For example, the dissociating means can include physical and/or a chemical disruption. Physical dissociation means can include, for example, scraping the tissue sample with a scalpel, mincing the tissue, physically cutting the layers apart, or perfusing the tissue. Chemical dissociation means can include, for example, digestion with enzymes disclosed herein or used in the art. Chemical dissociation can include the use of more than one enzyme and can include the use of two, three, or four enzymes. In one aspect, when more than one enzyme is used the enzymes are used sequentially. In one aspect, when more than one enzyme is used, they are selected from gentlyase, trypsin, dispase, and collagenase. Non-enzymatic solutions for the dissociation of tissue can also be used. Dissociation of the tissue sample can be achieved by placing the sample in a pre-warmed enzyme solution containing an amount of proteolytic enzyme sufficient to dissociate cellular stratum in the tissue sample.

[0043] In an example, a non-autologous cell suspension can be used to produce cells capable of reproduction for purposes of skin grafting. One of ordinary skill in the art will appreciate that as used here skin grafting means using skin cells derived from skin, not transferring a biopsied piece of skin directly to a wound or injury site. To procure cells of any source, the cells can be suspended in an aqueous saline/nutrition solution. The solution can be anything physiological from a basic salt solution to a more complex nutrient solution. In an example, the nutrient solution can be free of all serum but contain various salts, such as electrolytes, which resemble the substances found in body fluids. This type of solution can be referred to as physiological saline. [0044] Phosphate or other non-toxic substances can also buffer the solution to maintain the pH at approximate physiological levels. Suitable nutrient solutions can be based on Ringer-lactate solutions, including, but not limited to, Hartmann's solution, dialysis solutions, and on peripheral intravenous nutrition solutions.

[0045] Whether using autologous or non-autologous sources, the volume of solution applied to the tissue sample after harvesting, or by suspending nonll autologous cells, can be small, otherwise the suspension may become too fluid, therein presenting difficulties in applying the suspension to the graft site. The actual volume of solution applied can depend, in part, on a preference of the healthcare practitioner or needs of the patient.

[0046] The composition of cells in the cellular preparation can be comparable to that seen in situ in other cellular preparations such as the cultured epithelial autograft (CEA) method. The composition of the cells in the cellular preparation described herein can contain the basal keratinocytes and skin progenitor cells for skin regeneration, which can typically be lost in the CEA method. Whereas conventional methods lose cellular constituents, such as skin progenitor cells, because of selective culture for keratinocytes, the cellular suspension described herein can have a cell composition comparable to the in situ cell population.

[0047] In an example, the reagents and solutions used with the cells can be sterilized before being mixed with the cells and forming the treatment solution. In one aspect, the reagents and solutions are sterile when obtained.

[0048] Upon isolation of cells using the system and methods of the invention, the cell can be used or administered in numerous ways. In one aspect, the cells are administered to a subject in need thereof. Administration can be, for example, topical, direct, intravenous, intra-arterial, oral, buccal, ophthalmic, intra-nasal, aerosol, and done by any reasonable means, including use of a syringe, an applicator, a pipette, an eye dropper, or a spray device. In one aspect, the isolated cells are used in vitro, such as in establishing a primary culture. When used in vivo for therapy, the isolated cells of the subject invention are administered to the subject in therapeutically effective amounts (i.e., amounts that have the desired therapeutic effect).

[0049] Cells of the invention can be administered to a subject in various ways for various purposes. Additional therapeutic agents and additional ingredients may also be administered with the cells, depending on how the cells are administered and for what purpose. The additional therapeutic agents and additional ingredients include, but not limited to, drugs, anti-inflammatory agents, anti-microbial agents, and use of pharmaceutical compositions comprising cells useful for treatment of the diseases disclosed herein as an active component or ingredient. Such a pharmaceutical composition may consist of the cells alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the cells and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The additional ingredients may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art. As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

[0050] Although the descriptions of isolated cells provided herein are principally directed to pharmaceutical compositions, saline solutions, or solutions as described herein comprising those cells which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

[0051] Another embodiment provides a method to prevent or treat a disease or condition comprising administering to a subject in need thereof an effective amount of a solution or pharmaceutical composition comprising an effective amount of isolated cells of the invention, and optionally additional agents or ingredients. [0052] The present invention further provides a kit comprising the system and device as well as one or more reagents. The present invention further provides an instructional material for the use thereof.

[0053] Reference is made to U.S. Application Serial No. 13/573,003, filed on August 13, 2012, entitled “DEVICE FOR CELL SPRAYING, MANUFACTURING OF THE DEVICE, METHOD FOR SPRAYING WITH THE DEVICE AND A CELL SUSPENSION SPRAYED WITH THE

DEVICE”, issued as U.S. Patent No. 9,505,000, and U.S. Application Serial No. 14/136,681, filed on December 20, 2013, entitled “CELL SPRAYING DEVICE, METHOD AND SPRAYED CELL SUSPENSION”, issued as U.S. Patent No. 9,610,430, and U.S. Pat. No. US 10,376,658 which issued on August 13, 2019, PCT Patent Application No. PCT/US2017/037274, PCT Patent Application No. PCT/EP2017064094, and German Patent No. DE102011100450B4, all of which are incorporated by reference herein in their entirety.

[0054] This overview is intended to provide an overview of subject matter of the present patent application. It is not intended to provide an exclusive or exhaustive explanation of the invention. The detailed description is included to provide further information about the present patent application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document.

[0056] FIG. 1. la is a schematic sectional view of an exemplary cell isolation device, including its subunits.

[0057] FIG. 1. lb is a schematic sectional view of another exemplary cell isolation device.

[0058] FIG. 1.1c is a schematic sectional view of another exemplary cell isolation device.

[0059] FIG. 1. Id is a photographic image of an exemplary cell isolation device in a partially disassembled state. [0060] FIG. 1.2a is a cross sectional view of an end of a cell isolation device.

[0061] FIG. 1.2b is a cross sectional view of an end of a cell isolation device.

[0062] FIG. 1.3a is a bottom perspective view of a tissue digestion chamber. [0063] FIG. 1.3b is a side view of the tissue digestion chamber of FIG. 1.3a. [0064] FIG. 1.3c is a cross sectional view of an inside of the tissue digestion chamber of FIG. 1.3 a.

[0065] FIG. 1.3d is a sectional top view of the tissue digestion chamber of

FIG. 1.3 a.

[0066] FIG. 1.3e is a sectional side view of a cell isolation device.

[0067] FIG. 2a is a perspective view of a biopsy holder in an open position.

[0068] FIG. 2b is a perspective view of the biopsy holder of FIG. 2a in a closed position.

[0069] FIG. 3 is a cross-sectional schematic of a cell sieve unit of the cell isolation device.

[0070] FIG. 4a is an image of another cell isolation device in a partially assembled position.

[0071] FIGS. 4b and 4c are additional images of the cell isolation device of FIG. 4a.

[0072] FIG. 4d is a representative schematic of the cell isolation device of FIGS. 4a-4c.

[0073] FIG. 5a is a side view schematic of another cell isolation device. [0074] FIG. 5b is another side view of the cell isolation device of FIG. 5a.

[0075] FIG. 5c is a top perspective view of the cell isolation device of FIGS.

5a and 5b.

[0076] FIGS. 6a and 6b are side view schematics of the cell isolation device of FIGS. 5a-5c.

[0077] FIG. 6c is a cross-sectional view of the device of FIGS. 6a and 6b.

[0078] FIG. 7 is an image of a waste filter membrane for use in the cell isolation device.

[0079] FIG. 8 is a cross-sectional schematic of a cell sieve unit and waste filter membrane of the cell isolation device. [0080] FIG. 9 is a perspective view of a schematic of a portion of a cell isolation device with an outer cover of the device removed.

[0081] FIG. 10 is a perspective view of a schematic of a portion of another cell isolation device.

[0082] FIGS. 11 and 12 are perspective views of schematics of filter tests chambers of two cell isolation devices.

[0083] FIG. 13 is an image of another cell isolation device.

[0084] FIG. 14a is an image a portion of a cell isolation device, including a biopsy holder.

[0085] FIG. 14b is an image of an epidermal-dermal separation of a skin biopsy after the digestion process using the biopsy holder of FIG. 14a.

[0086] FIG. 15a is an imagine a portion of a cell isolation device, including an air bubbling system.

[0087] FIG. 15b is an imagine of a cell isolation device, including an air bubbling system.

[0088] FIG. 16 is a schematic illustrating a cell isolation enzyme principle.

[0089] FIGS. 17a-17c are schematics illustrating a cell isolation process.

[0090] FIG. 18 shows images from part of a process for separating skin layers in a cell isolation device.

[0091] FIG. 19 is an expanded view schematic of another cell isolation device.

[0092] FIG. 20 is a schematic of a waste filter membrane for use in a cell isolation device.

[0093] FIG. 21 is a schematic of a cell sieve unit incorporating the waste filter membrane of FIG. 20.

[0094] FIGS. 22a-22c are photographic images of foam development in the cell digestion chamber at different air flow rates.

[0095] FIG. 23a-23d are images of anti-foam structures for use in a cell isolation device.

[0096] FIG. 24 is an expanded view schematic of a tissue digestion chamber with an air burst filter and an anti-foam lid.

[0097] FIG. 25 is an expanded view schematic of another tissue digestion chamber with anti-foam structures. [0098] FIG. 26 is an image of testing a tissue digestion chamber with anti- foam structures.

[0099] FIGS. 27a-27d are photographic images of foam development in the tissue digestion chamber using different anti-foam structures with trypsin.

[00100] FIGS. 28a-28d are photographic images of foam development in the tissue digestion chamber using different anti-foam structures with collagenase.

[00101] FIG. 29 is a schematic view of a system including three separate units and other components.

[00102] FIG. 29a is a schematic view of another embodiment of the system of FIG. 29 having a parallel arrangement of multiple waste filter units.

[00103] FIG. 30 is a perspective view of a tissue digestion chamber according to one embodiment.

[00104] FIG. 30a is an enlarged view of a portion of the tissue digestion chamber of FIG. 30.

[00105] FIG. 30b is a cross-sectional view of the portion of the tissue digestion chamber of FIG. 30a.

[00106] FIG. 31 is a cross-sectional view of a portion of a tissue digestion chamber according to another embodiment.

[00107] FIG. 32 is a perspective view of a cell sieve unit according to one embodiment.

[00108] FIG. 32a is a cross-sectional view of the cell sieve unit of FIG. 32.

[00109] FIG. 33 is an exploded view of a waste filter membrane unit according to one embodiment.

[00110] FIG. 34 is a perspective view of the waste filter membrane unit of FIG. 33.

[00111] FIGS. 35-42 show a process of cell concentration and waste filtering using the waste filter membrane unit of FIGS. 33 and 34.

[00112] FIG. 43 is a cross-sectional view of a waste filter membrane unit having a displacement body according to another embodiment.

[00113] FIG. 44 is a perspective view of a waste filter membrane unit having one or more spacers according to another embodiment.

[00114] FIG. 44a is an enlarged perspective view of the waste filter membrane unit of FIG. 44 with a cover removed. DETAILED DESCRIPTION

[00115] The present application is directed to a cell isolation device to isolate cells from tissues. The cell isolation device is a point of care medical device. It can be used in multiple settings or locations, such as in an operating room or an adjacent room to isolate cells mechanically and enzymatically from a tissue extracted from a subject’s body. In an example, the cells can be skin cells. The isolated cells can be used, for example, in a cell spray device to deliver the cells to a wound, such as a burn. The present application includes multiple examples of cell isolation devices with varying features and functionality and multiple processes used in isolating cells with the cell isolation device.

[00116] Abbreviations and Acronyms:

[00117] CEA - cultured epithelial autograft

[00118] CID - cell isolation device

[00119] DPBS - Dulbecco's phosphate-buffered saline solution

[00120] DSST - Donor Site Skin Tissue

[00121] LRS - Lactated Ringer’s Solution

[00122] STSG - split-thickness skin grafts

[00123] U/ml - units per milliliter

[00124] Definitions:

[00125] In the event of inconsistent usages between this document and any documents so incorporated by reference, the usage in this document controls. In this document, the articles “a” or “an” are used, as is common in patent documents, to include one or more than one, independent of any other instances or usages of “at least one” or “one or more.” By way of example, "an element" means one element or more than one element.

[00126] The term "about," as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. For example, in one aspect, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%. [00127] The terms "additional therapeutically active compound" or "additional therapeutic agent," as used in the context of the present invention, refers to the use or administration of a compound for an additional therapeutic use for a particular injury, disease, or disorder being treated. Such a compound, for example, could include one being used to treat an unrelated disease or disorder, or a disease or disorder which may not be responsive to the primary treatment for the injury, disease or disorder being treated.

[00128] The terms “aggregate”, “cluster”, “conglomerate” or variations thereof are used interchangeably herein.

[00129] As used herein, "alleviating a disease or disorder symptom," means reducing the severity of the symptom or the frequency with which such a symptom is experienced by a patient, or both. A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

[00130] The term "biocompatible," as used herein, refers to a material that does not elicit a substantial detrimental response in the host.

[00131] The term "biodegradable," as used herein, means capable of being biologically decomposed. A biodegradable material differs from a non- biodegradable material in that a biodegradable material can be biologically decomposed into units which may be either removed from the biological system and/or chemically incorporated into the biological system.

[00132] The term "biological sample," as used herein, refers to samples obtained from a subject, including, but not limited to, skin, hair, tissue, blood, plasma, cells, sweat and urine.

[00133] As used herein "burn" or "burns" refer to any detectable injury to tissue caused by energy applied to the tissue. The terms "burn" or "burns" further refer to any burning, or charring of the tissue, including thermal burns caused by contact with flames, hot liquids, hot surfaces, and other sources of high heat as well as steam, chemical burns, radiation, and electrical burns. First degree burns show redness; second degree burns show vesication; third degree burns show necrosis through the entire skin. Burns of the first and second degree are partial-thickness burns, those of the third degree are full-thickness burns. [00134] The terms "cell culture" and "culture," as used herein, refer to the maintenance of cells in an artificial, in vitro environment. It is to be understood, however, that the term "cell culture" is a generic term and may be used to encompass the cultivation not only of individual cells, but also of tissues, organs, organ systems or whole organisms, for which the terms "tissue culture," "organ culture," "organ system culture" or "organotypic culture" may occasionally be used interchangeably with the term "cell culture."

[00135] The phrases "cell culture medium," "culture medium" (plural "media" in each case) and "medium formulation" refer to a nutritive solution for cultivating cells and may be used interchangeably.

[00136] A "compound," as used herein, refers to any type of substance or agent that is commonly considered a drug, or a candidate for use as a drug, combinations, and mixtures of the above, as well as polypeptides and antibodies of the invention.

[00137] A "conditioned medium" is one prepared by culturing a first population of cells or tissue in a medium, and then harvesting the medium. The conditioned medium (along with anything secreted into the medium by the cells) may then be used to support the growth or differentiation of a second population of cells.

[00138] A tissue "normally comprises" a cell if one or more of the cells are present in the tissue in an animal not afflicted with a disease or disorder.

[00139] The term "delivery vehicle" or "second device" refers to any kind of device or material which can be used to deliver cells in vivo or can be added to a composition comprising cells administered to an animal. This includes, but is not limited to, implantable devices, syringes, matrix materials, scaffold materials, gels, etc.

[00140] As used herein, an "effective amount" or "therapeutically effective amount" means an amount sufficient to produce a selected effect, such as alleviating symptoms of a disease or disorder. In the context of administering compounds in the form of a combination, such as multiple compounds, the amount of each compound, when administered in combination with another compound(s), may be different from when that compound is administered alone. Thus, an effective amount of a combination of compounds refers collectively to the combination, although the actual amounts of each compound may vary. The term "more effective" means that the selected effect is alleviated to a greater extent by one treatment relative to the second treatment to which it is being compared.

[00141] "Graft" refers to any free (unattached) cell, tissue, or organ for transplantation.

[00142] "Allograft" refers to a transplanted cell, tissue, or organ derived from a different animal of the same species, while “Autograft” refers to a graft of tissue from one point to another of the same individual's body.

[00143] "Xenograft" refers to a transplanted cell, tissue, or organ derived from an animal of a different species.

[00144] The term "improved blood flow," as used herein, refers to increased blood flow in a subject being treated according to the methods of the invention compared with the flow in a subject with an otherwise identical injury or condition not being treated according to the methods of the invention. Improved flow is determined by methods such as those described herein and can include less stasis, less sludging, or a combination of both, in the subject being treated compared with the untreated subject.

[00145] In this document, the terms “including” and “in which” are used as the plain-English equivalents of the respective terms “comprising” and “wherein.” The terms “including” and “comprising” are open-ended, that is, a system, device, article, composition, formulation, or process that includes elements in addition to those listed after such a term in a claim are still deemed to fall within the scope of that claim. Moreover, in the context of claims, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects.

[00146] The term "injury" refers to any physical damage to the body caused by violence, accident, trauma, or fracture, etc., as well as damage by surgery.

[00147] As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the identified compound invention or be shipped together with a container which contains the identified compound.

Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

[00148] The term "material" refers to any compound, molecule, substance, metal, alloy, or group or combination thereof that forms any type of structure or group of structures for use in making or using the devices and system of the present application.

[00149] The terms “insert” or “inserting”, as used herein and in reference to the cell isolation device, describe introducing one or more liquids, substances, materials, etc. into the cell isolation device or a component thereof. For example, “inserting enzymes into the tissue digestion chamber” means pumping enzymes into the tissue digestion chamber using an enzyme solution as described herein.

[00150] In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated. A "sample," as used herein, refers preferably to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, or fluid of interest. A sample can also be obtained from cell or tissue culture.

[00151] A "subject" of diagnosis or treatment is an animal, including a human. It also includes pets and livestock.

[00152] As used herein, a "subject in need thereof is a patient, animal, mammal, or human, who will benefit from the method of this invention.

[00153] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs. [00154] A "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

[00155] The term "thermal injury" is used interchangeably with "thermal burn" herein.

[00156] "Tissue" means (1) a group of similar cells united to perform a specific function; (2) a part of an organism consisting of an aggregate of cells having a similar structure and function; or (3) a grouping of cells that are similarly characterized by their structure and function, such as muscle or nerve tissue.

[00157] The term "topical application," as used herein, refers to administration to a surface, such as the skin. This term is used interchangeably with "cutaneous application" in the case of skin. A "topical application" is a "direct application".

[00158] The term to "treat," as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the frequency with which symptoms are experienced.

[00159] A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[00160] Cell Isolation Device:

[00161] Disclosed herein are multiple examples and designs of cell isolation devices that can be used to isolate cells from tissue.

[00162] FIG. 1. la is a schematic representation (sectional view) of an exemplary cell isolation device 100, including it subunits, the tissue digestion chamber 102, the biopsy holder 104, and the cell sieve unit (or filter unit) 106. In another example, there is no biopsy holder included in the cell isolation device 100. The cell isolation device 100 can include multiple inlets and outlets, which can be used for input of washing solutions, waste/output of wash solutions, and an opening to a filtration unit where cells leave the digestion core or digestion chamber. In an example, the cell sieve unit 106 has three separate filters 108 A, 108B and 108C, and each filter can have a different pore size. [00163] As it is shown in FIG. 1.1a, the cell isolation device 100 can be subdivided into three main parts, the tissue digestion chamber 102 (L x D x H: 131 x 35.5 x 72 mm), the biopsy holder 104, and the cell sieve (filter) unit 106 (L: 131 mm and 0 42.3 mm). Its principal function is to allow the mechanical tissue separation, afterward digestion, cell suspension, and washing steps while maintaining the temperature at 37°C.

[00164] In an example, the tissue digestion chamber 102 is a 50 mL volume capacity cylindrical unit comprising three ports for air/liquid interchange: (1) an inlet port 110A that allows pumping enzymes and saline solution into the chamber, (2) an air vent valve 110B to keep the correct inner pressure, and (3) an outlet port 110C to evacuate waste products.

[00165] During the tissue digestion process, the inlet port 110A allows filling of the tissue digestion chamber by pumping an enzyme solution such as enzyme/LRS while the air vent valve 110B keeps the inner pressure constant. The waste outlet port 110C allows the emptying process of the tissue digestion chamber by pumping out the used enzymes or the LRS cell washes.

[00166] In the main body of the tissue digestion chamber the biopsy holder 104 can be inserted, which in turns keep the tissue biopsy in place and secured during the tissue digestion process (see cross section in FIGS. 1.2a and 1.2b). The biopsy holder 104 is described further below.

[00167] In another aspect, the cell isolation device 100 comprises multiple inlets and at least one waste outlet and is connected to a filter housing unit comprising filters (membrane). Multiple waste outlets can allow for a better distribution of the liquid and improve flushing of a waste solution.

[00168] In one aspect, the waste outlet 110C of the tissue digestion chamber has a 5.0 pm pore-size membrane to allow fluid interchange without losing the isolated cells (see, for example, FIG. 1.3e and described below). Of course, the pore size can vary, and more than one filter can be fitted into the waste outlet if needed, including filters with varied pore sizes. A primary function of the tissue digestion chamber 102 includes providing housing for the biopsy holder 104, enzyme interchange, cell washing steps with a saline solution, and to maintain the cells under an isotonic solution until they are harvested into a second device (e.g., a syringe or other container as discussed herein). In one aspect, the tissue digestion chamber 102 has two or more waste outlets.

[00169] FIG. 1.1b is a sectional view schematically representing an exemplary cell isolation device 100A with a chamber 101 A connecting the filter/sieve unit (on the left) with the tissue digestion chamber and biopsy holder (on the right).

[00170] FIG. 1.1c is a sectional view schematically representing another cell isolation device 100B having a mechanism 10 IB to open or close a channel between the filter/sieve unit on the left with the rest of the device on the right. [00171] FIG. 1. Id is an image of an exemplary cell isolation device 100C in a partially disassembled state. The cell isolation device of FIG. l. ld is similar to the design shown in FIG. 1.1c. FIG. l. ld shows the biopsy holder 106 removed from inside the tissue digestion chamber.

[00172] FIG. 1.2a is a schematic representation (cross section) of a cell isolation device 100D, including a biopsy holder 106D. FIG. 1.2a highlights small gaps (for example, about 0.75 mm) between a chamber housing 112D and a filter, the chamber housing 112D, a filter tube 114D, and the biopsy holder 106D. The filter tube 114D can be excluded in other examples.

[00173] FIG. 1.2b is a schematic representation (front section) of a cell isolation device 100E, showing a biopsy holder 106E but not the filter membrane. In an example, the tissue digestion chamber is a cylindrical unit with a 50 mL volume capacity that has three ports for air liquid interchange: an inlet port that allows pumping enzymes and saline solution (1), an air vent valve to maintain the desired inner pressure (2), and an outlet port to evacuate waste products (3). During the tissue digestion process, the inlet port allows for filling of the tissue digestion chamber by pumping the enzymes/LRS while the air valve port keep the inner pressure constant. The waste outlet port allows the emptying process of the tissue digestion chamber by pumping out the used enzymes or the LRS cell washes. In the main body of the tissue digestion chamber fits the biopsy holder 106E which in turn keeps the tissue biopsy secure during the tissue digestion process. A port 110E is indicated on the upper part of FIG. 1.2b. FIG. 1.2b also shows an example in which the filter is integrated into the tissue digestion chamber.

[00174] FIGS. 1.3a, b, c, and d are schematics of an exemplary cell isolation device 100F, and specifically show a tissue digestion chamber 102F with a focus on a waste outlet 110F and a filter membrane 116F. In an example, the waste outlet 110F of the tissue digestion chamber has about a 5 pm pore-size membrane to allow fluid interchange without losing the isolated cells. FIG. 1.3a illustrates a bottom view of a filter with a lid. FIG. 1.3b illustrates a side view and shows the outlet port. FIG. 1.3c is a section side view. FIG. 1.3d is a sectional top view with the filter membrane 116F exposed.

[00175] FIG. 1.3e is a sectional side view of another exemplary cell isolation device 100G. The tissue digestion chamber 102 of the device of FIG. 1.3e is similar to the chamber shown in FIGS. 1.3a-d. As shown in FIG. 1.3e, fluid tight sealing avoids dead spaces outside the filters. A cell sieve unit (or filter unit) 106 of the device includes three pluristrainer filters that are joined together, and there is a direct passage to the filters from the tissue digestion chamber. In the example shown in FIG. 1.3e, the digestion chamber 102 and filter housing (or cell sieve unit) 106 are connected as one single housing. In the example shown in FIG. 1.3e, a separate membrane filter 116G can be located in the tissue digestion chamber 102 (above the grid and waste outlet) to provide more filter surface and to reduce clogging of grid corners with debris. In one aspect, gaskets can be used where fittings occur between chambers or points of coupling. In one aspect, the tissue digestion chamber 102 provides housing to the biopsy holder (not shown), a site for enzyme interchange, the location for saline washing of cells, while maintaining the cells under isotonic conditions until harvesting into the second device. The left end of the digestion core has an opening/communication toward the filter unit 106 for delivery of cells in suspension to be filtered to remove debris and cell aggregates/clumps. In some embodiments, the filter unit 106 is housed within the same housing as the digestion core.

[00176] FIGS. 2a and 2b are schematics of an exemplary biopsy holder 104H for use in a tissue digestion chamber. The biopsy holder 104H can function to hold (contain), for example, a skin biopsy during its enzymatic digestion process, mechanical tissue separation, and wash steps. FIG. 2a shows the biopsy holder 104H in an open position and FIG. 2b shows the biopsy holder 104H in a closed position. The biopsy holder 104H can include an inner cylinder 118H and outer cylinder 120H. The biopsy can be rolled onto the inner cylinder 118H and then placed into the outer cylinder 120H. The outer cylinder 120H can contain holes for the enzymes to access the skin biopsy.

[00177] The outer cylinder 120H can be opened to insert the inner cylinder 118H (which contains the skin biopsy on the inner cylinder 118H) into the outer cylinder 120H. As illustrated in the example of FIGS. 2a and 2b, four snap-on supports 122H, 122HH, 122HHH and 122HHHH can be used to keep the skin biopsy secure while grooves on the outer case allow enzyme/saline solution interchange during the enzyme digestion and wash process. The configuration of the snap-on supports 122H, 122HH, 122HHH and 122HHHH can be varied, including more or less supports than the four shown in FIGS. 2a and 2b. Other types of attachment features can be used in addition to or as an alternative to the snap-on supports 122H, 122HH, 122HHH and 122HHHH shown in FIGS. 2a and 2b. The connections snap into place (snap-fit) when the biopsy holder 104H moves from the open to closed position. Also shown in FIG. 2a is a representation of a tissue biopsy in the biopsy holder. The grooves in the biopsy holder (labelled in FIG. 2b) allow for access to incise/cut the biopsied tissue.

[00178] Other biopsy holder designs can be included in the cell isolation devices disclosed herein. An alternative biopsy holder design is shown in the other FIGS, and described further below - see, for example, FIGS. 5c and 6c. [00179] After the tissue digestion in the chamber and the isolated cells are washed, the cell isolation device can be rotated approximately 90° to allow the cells in solution to move from the tissue digestion chamber to the cell sieve unit. During the sedimentation process, the isolated cells in solution can go through the sieves in the sieve unit to retain the larger particles while filtering the cells in solution.

[00180] FIG. 3 is a schematic cross section of an exemplary cell sieve unit 106. As shown in FIG. 1.3e, the cell sieve unit 106 can be connected and housed within the same unit as the tissue digestion chamber 102. FIG. 3 shows the cell sieve unit 106 after the cell isolation device has been rotated 90 degrees, as described above. In an example, the cell sieve unit 106 is a cylindrical case that contains three sieves 124, 126 and 128 with successively smaller pore sizes - for example, 200, 100 and 70 pm pore sizes, respectively - to sequentially pass cells, while excluding from the final cell solution tissue fibers, debris and large clusters (aggregates) of cells that might clog the filters while passing into the second device or clog a cell sprayer device. At the cell outlet are single cells or clustered cells (in solution) with a smaller size than 70 pm.

[00181] It is recognized that the cell sieve unit 106 can include more or less than the three sieves shown in FIG. 3. Moreover, the cell sieve unit 106 can include different pore size sieves than the 200, 100 and 70 pm pore sizes shown in FIG. 3. The sieves are used to sequentially isolate cells in the isolation chamber from fibers and clumps of cells, particularly large clumps. The number and types of sieves can depend in part on the type of tissue. In the case of using skin, clumps of cells and debris could clog the cell sprayer used to administer the cells to a wound such as a burn.

[00182] FIGS. 4a-4d are images of an exemplary cell isolation device 1001 with a chamber connection 1011 between the tissue digestion chamber 102 and the cell sieve unit 106. FIG. 4a illustrates the digestion chamber connected to the sieve unit 106 and adjacent to them are the biopsy holder 104 pieces, which have been removed from the digestion chamber 102. The cell isolation device 1001 can be modular. FIG. 4b illustrates how the biopsy holder 104 can be inserted into the digestion chamber 102 and FIG. 4c shows the digestion chamber 102 sealed with the biopsy holder 104 inside. FIG. 4d is another representation of the cell isolation device 1001 of FIGS. 4a-4c. The three sieves 124, 126 and 128 can be seen in FIG. 4d inside the cell sieve unit 106.

[00183] The cell isolation device 1001 in FIGS. 4a-4d is similar in function to the device shown in FIGS. 1.1c, 1. Id and 1.3e. The chamber connection 1011 between the tissue digestion chamber 102 and the cell sieve unit 106 can facilitate independent analysis of the two parts of the device, which can be helpful during development and testing. In an example, the chamber connection 1011 can be similar to a Luer lock connection.

[00184] FIG. 5a is a sectional side view of an exemplary cell isolation device 100 J that has similar functionality to the designs described above and some differences in specific design features. For example, the device of FIG. 5a includes a different biopsy holder 104 J design and a waste filter membrane 116J that is contained within the cell sieve unit 106J of the device. (The biopsy holder design 104J of FIG. 5a is further shown in FIGS. 5c, 6c, 14a, and 14b. The filter membrane design 116J of FIG. 5a is futher shown in FIGS. 5c, 6c, and 7-9.) Moreover, one or more waste outlets (two are shown in FIG. 5a) are contained within the cell sieve unit 106J rather than within the tissue digestion chamber 102J, as shown in the FIGS, described above. In the design shown in FIG. 5a, the waste outlets are closer to the port.

[00185] The device in FIG. 5a includes an air inlet and an enzyme/RL inlet, two air outlets and two waste outlets. It is recognized that more of less inlets and outlets can be included in other examples. The function of the enzyme/RL inlet and the waste outlets are generally similar to those described above in reference to the earlier FIGS.

[00186] FIG. 5b is a side view of the device of FIG. 5a rotated 180 degrees. FIG. 5c is a top perspective view of a schematic of the device of FIGS. 5a and 5b. A clear outer cover is used in FIG. 5c such that the inside components of the cell isolation device 100J are visible.

[00187] The cell isolation device 100J of FIGS. 5a-5c can include features, such as the air inlets and outlets shown in FIG. 5a, to facilitate an air bubbling system for tissue separation in the tissue digestion chamber. Such features can include, for example, an air inlet and two air outlets. The air bubbling system is described further below in reference to FIGS. 15a and 15b.

[00188] FIGS. 6a-6c are schematics of the cell isolation device 100J of FIGS. 5a-5c. FIG. 6c is a cross sectional view of the cell isolation device 100J and shows the biopsy holder 104 J, the three cell sieves 124, 126 and 128 and the waste filter membrane 116J. FIGS. 6a-6c show the cell isolation device 100J oriented vertically. As described further below, the design of the cell isolation device 100J in FIGS. 6a-6c (having a radially oriented filter membrane 116J near the port for the second device) allows for the cell isolation process to occur with the device oriented vertically. The radial waste filter membrane 116J can provide a sufficient filter surface area and help prevent clogging of the membrane. It is recognized that different sizes and shapes can be used for the waste filter membrane 116J. For example, the waste filter membrane 116J can be increased in size to increase the surface area for filtration. Moreover, the vertically oriented device of FIGS. 6a-6c is conducive to the air bubbling system used during cell isolation.

[00189] FIG. 7 is an image of only the waste filter membrane 116J that is shown near the port in the cell isolation device of FIGS. 5 and 6. The waste filter membrane 116J can also be referred to herein as filter membrane or membrane filter. In an example, the pore size of the waste filter membrane 116J can range between about 0.4 and 5 pm. In an example, the filter area can range between about 5.5 and 11 cm². It is recognized that any pore size membrane that is smaller than the last sieve can be used in the waste filter membrane 116J. In an example, the pore size of the waste filter membrane 116J is markedly smaller than the pore size of the last sieve - for example, 70 microns compared to 5 microns. A particular pore size selected can depend on the type of cells being isolated in the cell isolation device, as well as the pore size of the last cell sieve. In the example shown in FIG. 7, the waste filter membrane 116J can be cylindrically shaped and open on the top and bottom. The waste filter membrane 116J does not include a bottom filter - only filters arranged radially around sides of the cylindrical structure. This is described further below in reference to FIGS. 9-12.

[00190] FIG. 8 is a simplified schematic showing a cross section view of the cell sieve unit 106 J, including the three cell sieves or filters 124, 126 and 128, the filter membrane 116J, the waste port and the port. In the example of FIG. 8, three cell sieves 124, 126 and 128 are used, which function similar to the cell sieves described above in reference to FIG. 3. Again, more or less filters can be used in the cell sieve unit 106 J.

[00191] FIG. 9 illustrates the path of the cells and the solutions that can be flushed through the cell isolation device 100J. As provided above, the cell sieves are used to separate out larger debris and aggregates or clusters of cells. The solution exiting the last cell sieve can comprise substantially single cells and not aggregates of cells.

[00192] The solution exiting the last cell sieve can then flow into the waste filter membrane 116J and through the sides of the waste filter membrane 116J as solution waste and out of the cell isolation device 100J via the waste outlet or waste port. The cells from the solution cannot pass through the pores of the waste filter membrane 116J and thus remain inside the waste filter membrane 116J for collection. The design in FIG. 9 shows one waste outlet port. In other examples, the cell sieve unit 106 J can include two waste outlet ports that are on opposite sides of the unit (see, for example, FIGS. 6a-6c with left and right waste ports). The cells remain inside the unit and can reside in the cell collector of FIG. 9. When it is time to transfer the cells to a second device (e.g., syringe, etc. as described herein), the second device can be connected to the port and the cells can be transferred to the by suction. In one aspect, a container other than a syringe can be used by connecting the container to the port.

[00193] As shown in FIGS. 8 and 9, the waste filter membrane 116J can be located in proximity to the waste outlet port. It is recognized that the membrane filter can be located elsewhere in the device. In order to collect the cells inside the waste filter membrane 116J and the cell collector, saline can be flushed through the device. Although saline can commonly be used as one of the last steps in the cell isolation process, it is recognized that saline can also be flushed through the cell isolation device 100J at other stages of the cell isolation process. See, for example, FIGS. 17a-17c which provide an example of steps of the cell isolation process.

[00194] FIGS. 10-12 illustrate other examples of the cell sieve unit 106K or filter test chamber. Instead of outlet ports on the side and bottom of the cell sieve unit 106K, the devices in FIGS. 10-12 can include multiple outlet ports at the bottom of the device. FIGS. 11 and 12 illustrate different configurations and specifications for the cell sieve unit 106K and the waste filter membrane 116K. As shown in FIGS. 11 and 12, different filter areas and pore sizes can be used. Although FIGS. 10-12 show more examples of a cylindrical filter membrane 116K, it is recognized that the filter membrane 116K can have different shapes. [00195] FIG. 13 is an image of another cell isolation device 100L that can be similar in design to the cell isolation device of FIG. 9.

[00196] FIG. 14a is an image of a portion of a cell isolation device 100M with a focus on a biopsy holder 104M. FIG. 14b shows a skin biopsy 130 as it is being contained by the biopsy holder 104M and being digested. The biopsy holder 104M includes a metal rod 132 and clips 134 and 136 attached to the metal rod. The example biopsy holder 104M shown in FIG. 14b includes two clips 134 and 136, although it is recognized that more or less clips can be used. The skin biopsy 130 is cut in connected longitudinal strips (each approximately 4 mm wide). Then the skin biopsy 130 is placed vertically on the clips 134 and/or 136 (circular plastic cylinder holders) attached to the metal rod. In addition to or as an alternative to the clips 134 and 136 shown in FIG. 14b, other types of clips, clamps, rubber bands, or surgical sewing thread can be used to help hold the skin in place. Then the biopsy holder 104M can be placed inside the tissue digestion chamber and the screw lid can be used to close the device. [00197] Once the tissue digestion chamber is filled with the enzymes, the skin biopsy can flow freely, while being restrained by the clips 134 and/or 136 and exposed to the enzymes and bubbling effect. Compared to the cage-like structure of the biopsy holder in FIGS. 2a and 2b, where the skin is wrapped and subjected onto an inner cylindrical structure, the biopsy holder 104M of FIGS. 14a and 14b may provide more accessibility to the enzymes.

[00198] FIG. 15a is an image of a cell isolation device 100N configured for an air bubbling system 138 in the tissue digestion chamber 102N. The air bubbling system 138 is configured to enhance tissue disassociation and cell liberation by agitating the tissue exposed to specific enzymes. The air bubbling system 138 can include air inlet ports and air outlet ports. The device in FIGS. 6a-6c showed two air outlet ports at the top of the tissue digestion chamber, whereas FIG. 15a, as well as FIG. 15b, includes four air outlet ports at the top of the tissue digestion chamber 102N (and associated tubing). Similarly, the device in FIGS. 6a-6c includes two inlet ports for enzyme/saline/air at the bottom of the tissue digestion chamber, whereas FIGS. 15a and 15b include four inlet ports (two red, two white) at the bottom of the tissue digestion chamber 102N. Two inlet ports can be used for enzymes/saline and two inlet ports can be used for air. It is recognized that any number of inlet ports and outlet ports can be used to accommodate the different fluids circulating through the device or through one or more units of the device.

[00199] The tissue digestion chamber 102N is filled with the strips of skin biopsy (described above under biopsy holder) and attached by one end to the biopsy holder. The chamber is then filled through the Enzyme/Sahne/Air Inlet ports with saline solution or 37°C pre-warmed enzymes covering the skin tissue biopsy. Enzyme/Saline/Air Inlet ports are versatile ports that allow the injection of liquids and air into the tissue digestion chamber using pressurized air or peristaltic pumped air.

[00200] The air can be continuously injected into the tissue digestion chamber 102N or the air can be pulsed at any interval or variably pulsed, or a mix of both. For example, pulsing can range from one pulse per second to one pulse per minute. In another example, one pulse can be delivered every two seconds for five minutes, then one pulse per minute for ten minutes, and then one pulse per second for 5 minutes. In an example, the air is injected at 50-300 mL/min airflow into the tissue digestion chamber through the Enzyme/Saline/Air Inlet ports and collected through the Air Outlet Port to balance the inner pressure. A specific rate can depend, in part, on the type of enzyme. Collagenase can create more foam compared to trypsin or gentlyase. The injected air is filtered first using a 0.22-0.45 pm air filter to obtain sterile air before pumping the air into the tissue digestion chamber.

[00201] The injected air produces around 0.5 cm diameter air bubbles from the right and left port and from bottom to top. In another example, the air bubbling system 138 can use central air inflow combined with a distributor. The airflow produces bubbles that continuously create a symmetric liquid displacement, which, in turn, moves the skin biopsy while it has been digested by the enzymes. During the tissue digestion process, the specific enzymes can target the proteins separating the epidermis for the dermis or liberating the cells from the adjacent tissue. (See FIG. 16.) The bubble effect can provide enough kinetic force to facilitate cell liberation.

[00202] After each enzyme digestion step, the enzyme solution can be aspirated and the skin biopsy can be rinsed with saline solution to wash any traces of the enzymes. During this washing step, the bubbling system can help to liberate the remaining cells from the adjacent tissue without enzyme presence. [00203] The air bubbling system 138 can be used as an alternative to the shaking/agitation step described for other configurations of the cell isolation device. In reference to the cell isolation devices of FIGS. 1.3a-e and FIG. 3, the process for cell isolation can include an initially horizontal orientation of the device and then rotation of the device 90 degrees such that the cell isolation device is oriented vertically. Once vertically oriented, the device can be shaken to agitate or disrupt the tissue. In an example, these steps can be repeated. (This is described further below.) The air bubbling system 138 can replace such shaking steps or be used in addition to shaking.

[00204] FIG. 15b illustrates a cell isolation device 100N prototype having a tissue digestion chamber 102N that is configured similar to the tissue digestion chamber 102N of FIG. 15a, including 4 inlet ports and four air outlet ports and associated tubing. The cell isolation device 102N includes a cell sieve unit 106N that is similar to the cell sieve units shown in FIGS. 10-12. In an example, and as shown in FIG. 15b, the device can include two outlet ports in the cell sieve unit - a waste outlet port and a port to collect the isolated cells. In other examples, additional outlet ports can be included in the cell sieve unit - for example, two waste ports.

[00205] The cell isolation device of FIG. 15b is a prototype and is shown as having a connection 10 IN between a tissue digestion chamber 102N and a separate cell sieve unit 106N. This is primarily so that the tissue digestion chamber 102N and the cell sieve unit 106N can be tested separately and independently of one another. For example, FIG. 15a shows just the tissue digestion chamber 102N with the connector 10 IN (FIG. 15b) capped off so that testing and evaluation can be done on the tissue digestion chamber 102N. In another example, the cell isolation device 102N can be configured as a single tissue digestion chamber/cell sieve unit that does not require a connection, as seen in other FIGS. In such one-piece unit or design, a valve can be used to control flow between the tissue digestion chamber 102N and cell sieve unit 106N. See, for example, FIG. 19.

[00206] Accessories:

[00207] The automated cell isolation device can use a wide range of enzymes to digest a diverse range of tissues and it can be done in combination with mechanical vibration or shaking to agitate or disrupt the tissue. For the epidermal and dermal cell isolation process, three different enzymes were used in a sequential process alternating the tissue digestion with saline solution rinses and washes. The enzymes and washing solutions used during skin cell isolation processes are described in the following section:

[00208] Enzyme Solutions

[00209] Dispase II

[00210] Dispase II (Roche) is a proteolytic enzyme derived from Bacillus polymyxa and used for isolating cells from tissue. Based on the manufacturer's labelling, dispase is gentler on cells than other proteolytic enzymes. Dispase II is provided by the supplier as a lyophilized powder. It is reconstituted in LRS prior to storage and use. The recommended concentration of dispase solution for use in epidermal and dermal tissue separation is 2.4 units per mL (U/mL). Because the unit concentration of powder may vary among the different product lots, calculations that ensure the correct concentration in the prepared solution are provided. The lyophilized powder is stable at 2-8°C until the expiration date printed in the label. Stock solutions are filtered with a 0.22 pm pore with low protein-binding membranes (e.g., materials like cellulose acetate, PVDF, or PES) and stored frozen at -15 to -25°C in aliquots for no longer than 6 months. Re-freezing enzyme solutions after thawing should be avoided. Because of its bacterial origin, dispase II is free of animal viruses and mycoplasmas. Dispase has proven to be an effective, fast-acting enzyme for the separation of epidermal and dermal tissues, while maintaining cell viability.

[00211] Trypsin-EDTA

[00212] Trypsin-EDTA (Thermofisher) is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation. Trypsin-EDTA is supplied as an enzyme solution, diluted in Hank's Balanced Salt Solution in 100 mL units. Processing this product requires transfer into 20 mL aliquots. The shelf life of Trypsin-EDTA is 24 months from the date of manufacture. It should be stored frozen at -15 to -20°C. Avoid exposing the product to direct sunlight. Trypsin- EDTA is manufactured at a cGMP compliant facility that is registered with the FDA as a medical device manufacturer and is certified to ISO 13485 standards. Trypsin-EDTA solutions are tested for pH, osmolality, sterility, and performance. In addition, raw materials used in manufacture are verified for e- beam irradiation and tested for endotoxin, PPV, PCV 1/2, mycoplasma, bacterial, fungal, and viral contamination, as well as multiple activity assays, ash analysis, and moisture analysis. Another type of trypsin that can be used, for example, is Trypsin-GMP (Roche).

[00213] Collagenase NB6

[00214] Collagenase NB6 (Nordmark Arzneimittel) is designed for dissociation of different tissues to isolate various cell types, including human fibroblasts from skin. Collagenase depends on calcium ions; meaning that > 2 mM Ca²⁺ should be included in the enzyme solution, and no calcium chelating agents (e.g. EDTA) should be used. The solvent Dulbecco's phosphate-buffered saline solution (DPBS) contains 0.133 mg/ml Ca²⁺, sufficient for adequate enzymatic activity. Stock solution is diluted with dissociation buffer to achieve the required enzymatic activity of 0.4 PZ-U/ml.

[00215] Collagenase NB6 is provided as a lyophilized powder and should be stored in a dry environment at 2-8 °C for 5 years. Stock solutions are filtered with a 0.22 pm pore with low protein-binding membranes (e.g., materials like cellulose acetate, PVDF, or PES) and stored frozen at -20 °C in aliquots for no longer than one year. Re-freezing enzyme solutions after thawing should be avoided. Another type of collagenase that can be used, for example, is Collagenase I-GMP (Roche).

[00216] Gently ase

[00217] Instead of Dispase II, GentLyase-GMP (a neutral protease) can be used. Although a process utilizing three enzymes is described above, it is recognized that a cell isolation process can alternatively use two of the three enzymes above - for example, GentLyase and Collagenase I.

[00218] All enzymes are handled and stored according to instructions for that particular enzyme.

[00219] Wash Solutions

[00220] Lactated Ringer's solution

[00221] Lactated Ringer's solution (LRS) (Baxter) is an aqueous solution that provides an osmotically balanced environment and is capable of keeping isolated cells alive up to 24 hours. LRS is sterile, non-pyrogenic, and contains no bacteriostatic or antimicrobial agents. The LRS product is provided in a 1,000 mL plastic pour bottle container. It is shipped at room temperature with a shed life of 36 months from the date of manufacture. Store the product at 25°C without exposure to direct sunlight.

[00222] Dulbecco's phosphate-buffered saline

[00223] Dulbecco's phosphate-buffered saline solution (DPBS) (Thermofisher) is a balanced salt solution used for a variety of cell culture applications. Its main role in cell culture is maintaining intra- and extra-cellular osmotic balance, acting as a buffering system to maintain the medium in a physiological pH range. It provides cells with water and certain bulk inorganic ions essential for normal cell metabolism.

[00224] DPBS is shipped at room temperature and the shelf life is 36 months from the date of manufacture. It is recommended to store the product at 15-30°C without exposure to direct sunlight. Twenty -four hours prior to performing enzyme preparation steps below, transfer DPBS to refrigerated storage (4°C). [00225] In an example, the Cell Isolation Device uses a sequential three step enzyme digestion to make accessible the tissue regions combined with mechanical tissue vibration to optimize the cell isolation harvest. In an example, mechanical vibration can include shaking. In an example, mechanical vibration can include an air bubbling system. In other examples, a combination of shaking and air bubbling can be used for vibration.

[00226] FIG. 16 illustrates use of three enzymes - Trypsin, GentLyase and Collagenase - for separating the tissue layers and liberating specific cells. Trypsin liberates epidermal cells, including stem cells, keratinocytes, melanocytes and other epidermal cell types. Gentlyase separates dermis from epidermis and exposes epidermal stem cells in the basal layer to action by Trypsin while exposing the dermal fibroblasts and stem cells of papillary dermis to Collagenase. Collagenase liberates dermal progenitor cells and stem cells from the dermis layer. In another example, Dispase II can be used instead of Gentlyase.

[00227] FIGS. 17a-17c provide a cell isolation process 10 using three enzymes and the air bubbling system (or shaking) in the tissue digestion chamber described above. The steps labelled in FIGS. 17a-17c correspond with the steps in Tables 1 and 2 below. [00228] Cell Isolation Process (using shaking):

[00229] The process 10 description below can use, for example, the cell isolation device and components shown in FIGS. 1.3a-e, 2a-2b and 3. Such process can include rotation of the device between a horizontal and vertical orientation. A similar process can be used for the cell isolation devices of FIGS. 5-15; however, rotation of the device is excluded from that process since the devices of FIGS. 5-15 are configured for vertical orientation and incorporate the air bubbling effect described above. It is recognized that the process can include more or less wash steps compared to what is shown in Table 1 below.

Table 1 : Process steps with shaking and rotation

[00230] The volume and time/duration and flows of each step, including pumping in/shaking/pumping out, can be defined as well as the total time for the cell isolation process until the removal of the filled syringe for the cell spray application. All or substantially all of the tissue digestion chamber is filled and emptied between 60 seconds and 5 minutes (Flow rate approximately 0.1 to_4 ml/s).

[00231] Cell Isolation process (using air bubbling):

[00232] The process description below can use, for example, the cell isolation device and components shown in FIGS. 5a-5c; 6a-6b, 15a-15b. Such process can include the provision of air via injection, peristaltic pump, or others. A similar process can be used for the cell isolation devices of FIGS. 5-19.

Table 2: Process steps with air bubbling

[00233] In the process steps of Table 2 above, air can be injected continuously or via pulsing. The injection can depend at least on the type of enzyme in the tissue digestion chamber. For example, continuous air may be used with trypsin whereas pulsing air may be used with collagenase.

[00234] Potential use of anti-foam agents or structural modifications/additions can be used to eliminate foam formation during the air bubbling steps. These are described further below and shown in FIGS. 23-28.

[00235] Parameter description (using shaking and/or bubbling):

[00236] The process starts with loading the biopsy holder with skin biopsy harvesting using an electrical dermatome. The optimal skin thickness is 8/100 inch (0.02 cm.) composed of the entire epidermal tissue fraction and the upper dermal tissue fraction. The biopsy holder has holes or slots to allow making incisions into the skin biopsy, thus enabling the enzyme to penetrate into the tissue.

[00237] Another step is to pump Dispase II into the tissue digestion/cell isolation chamber though the enzyme/LRS inlet port. At the same time, the air valve compensates the inner chamber overpressure by releasing the air outside (FIGS. 1.1 and 1.3). The 40 mL Dispase II enzyme can penetrate the biopsy holder through the grooves (FIGS. 2 and 4).

[00238] Tissue digestion and shaking/bubbling are another step. The digestion process can take about 40 minutes where the enzyme can penetrate into the skin tissue digesting the epidermal-dermal junction proteins. A shaking step can be applied every 10 minutes during this process to facilitate epiermal-dermal separation, exposing the stratum basale where the keratinocyte stem cells and rapidly-dividing keratinocyte reside. Alternatively, a continuous or pulsed air bubbling step (airflow of 100-300ml/min) can be added to facilitate epiermal- dermal separation. [00239] Waste can be pumped out. After the epidermal-dermal junction digestion and tissue separation has been completed, the 40 mL of Dispase II enzyme left can be pumped out by the wash outlet and through the membrane filter.

[00240] One step requires trypsin digestion and it can be pumped in following a waste pumping step. In one aspect, about 40 ml of trypsin is used. It is pumped into the tissue digestion chamber through the inlet port. It can take between 60 seconds and 5 minutes to fill in at a rate betweeen about 0.1 and 4 ml/s. Further digestion and shaking occurs at the trypsin step. For a time of about 15 minutes, trypsin can start digesting the stratum basale from the epidermal tissue, freeing and isolating keratinocyte stem cells and rapidly dividing keratinocytes from the tissue. A shaking step can be applied every 5 minutes during this process to facilitate epidermal cell isolation. As an alternative to or addition to shaking, air bubbling can be added to facilitate epidermal liberation from tissue. Air bubbling can be achieved by continuously introducing air into the tissue digestion chamber or pulsed air delivery (for example, airflow of 50-300 ml/min).

[00241] At that point another pumping of waste is done to pump out the used trypsin enzyme.

[00242] A large wash/volume of washing solution follows trypsin digestion. About 200 mL of LRS is pumped in to wash the cells while diluting any trace of trypsin enzyme left in the tissue digestion chamber. A variable volume of LRS can be left in the tissue digestion chamber to resuspend the cells (the volume can depend on the sprayable wound area).

[00243] Once the wash is complete, the cells are filtered out and aspirated as isolated cells into a syringe. The steps used to collect the cells into the syringe can depend on the particular cell isolation device.

[00244] Under a process which uses the device shown in FIGS. 1.3a-e, the tissue digestion chamber rotates 90° to allow cells in solution to settle by gravity (about 5-10 minutes). The cells in solution can then go through the series of cell sieves (for example, 200, 100 and 70 pm pore size) eliminating fibers, clumps, and debris from the final cell suspension. Then the cells can be aspirated from the cell isolation device to a syringe. [00245] Under a process which uses the device shown in FIGS. 5-15, the device does not need to be rotated in order to collect the cells. Rather, the cells in solution can flow into the cell sieve unit and go through the series of cell sieves. The cells can then be collected in the waste filter membrane and aspirated from the cell isolation device via a syringe.

[00246] Next, collagenase can be pumped into the tissue digestion chamber through the Inlet port (rate at 0.1 to 4ml/s). Digestion with shaking occurs and the collagenase can digest the dermal tissue for about 40 minutes. Alternatively or additionally, a continuous or pulsed air bubbling step can be added to facilitate the dermal liberation from tissue.

[00247] The fibroblasts and some dermal mesenchymal stromal like cells can be liberated into the chamber. A shaking step can be applied every 10 minutes during this process to facilitate dermal digestion.

[00248] The waste, about 40 mL of the used collagenase enzyme, can be pumped out.

[00249] Next, about 200 mL of LRS can be pumped in to wash the cells and to dilute any trace of collagenase enzyme left in the tissue digestion chamber. One milliliter of LRS can be left in the tissue digestion chamber to resuspend the cells.

[00250] At this point, cells can be obtained by aspirating the isolated cells into a syringe. The specific process steps at this point can depend on the design of the cell isolation device. As described above, for the device in FIGS. 1.3a-e, the tissue digestion chamber can rotate 90 degrees to allow cells in solution to settle by gravity (between 5-10 minutes), the cells in solution can then go through the cell sieves, eliminating fibers, clumps and debris from the final suspension, and then the cells in 1 mL of LRS can be aspirated from the tissue digestion chamber into the syringe with the already stored keratinocytes in solution.

[00251] For the device in FIGS. 5-15, rotation and settling by gravity is excluded since the device can be vertically oriented throughout the process. [00252] FIG. 18 illustrates testing of the tissue digestion chamber with the air bubbling system. As provided in Table 2 above, air bubbling can be used during dispase and trypsin digestion. [00253] Other designs for cell isolation device:

[00254] Several prior designs experienced clogging problems. Multiple prototypes have been designed and are disclosed herein to solve the clogging issue. For example, one potential issue resolved by the present application is where the enzyme/LRS, together with skin debris are flushed into the cell sieve unit but cannot be appropriately removed through the outlet port. Moreover, some amount of enzyme may enter the cell sieve unit and cannot be used during digestion (see FIGS.).

[00255] One prototype disclosed herein that resolves that issue is where the filter chamber is separated from the digestion chamber during the digestion process and they are then connected before the cell filtration begins (FIG. 1.1a, b, and c). Other prototypes are disclosed herein and the application encompasses others depending on performance testing during cell isolation.

[00256] For example, in one embodiment, a cell isolation device (Fig 1.1a) encompasses a solid one-piece prototype which offers the advantage of distributing the mechanical vibration to the whole structure, allowing for optimal tissue separation. On the other hand, the debris can potentially clog the cell sieve unit while losing enzyme volume in its space.

[00257] In another embodiment, another cell isolation device (Fig 1.1b) includes a physical separation between the two units allowing the communication between them only after the last wash step concluded. Then the cell suspension in the tissue digestion chamber can be transferred to the cell sieve unit by suction.

[00258] In a further embodiment, another cell isolation device (Fig 1.1c) is similar to the design in Fig 1.1a includes a magnetic valve or electromechanical valve that only opens after the last wash step has concluded. Then the cell suspension in the tissue digestion chamber can be transferred to the cell sieve unit.

[00259] FIG. 19 is a schematic with an expanded view of the various components of an example cell isolation device 1000. The cell isolation device 1000 of FIG. 19 is an example of a one-piece design, similar to FIG. 1.1c, and includes a feature (three-way stopcock 140) for controlling flow of fluids from the tissue digestion chamber 1020 to the filter unit or cell sieve unit 1060. This can allow tissue digestion to be separate from filtration. The three-way mechanically actuated stopcock of FIG. 9 is one example of the type of separation feature usable in the cell isolation device. A magnetically actuated stopcock can similarly be used. It is recognized that other types of features (whether manual or automated, battery operated, etc.) can be used to control the flow of fluids. In another example, a pinch valve can be used instead of the three-way stopcock of FIG. 19 to regulate solution flowing from the tissue digestion chamber to the filter unit/cell sieve unit.

[00260] In the design of FIG. 19, the air outlet ports can be part of the lid for the tissue digestion chamber 1020. In the design of FIG. 19, the filter unit/cell sieve unit 1060 includes two filters, (200 pm, 70 pm) rather than the three filters shown in other FIGS, and described above. It is recognized that more or less filters can be used in the filter unit. It is also recognized that the cell isolation devices can be modular and can be custom assembled to include different features described herein based on the user needs and specifications. Such modular design can facilitate, for example, custom assembly of specific filters in the cell sieve unit.

[00261] FIG. 20 is a schematic of another design of the waste filter membrane/cell sieve unit 106P/112P of the cell isolation device. Compared to the design in FIGS. 7, 11 and 12, the waste filter membrane 112P of FIG. 20 is horizontal. Rather than the sides of the waste filter membrane 112P having the filter material, the filter material forms the horizontal bottom of the waste filter membrane 112P in FIG. 20. The specifications of the waste filter membrane 112P are shown in FIG. 20. It is recognized that other iterations of this horizontal design can be used in addition to the specific parameters shown in FIG. 20.

[00262] FIG. 21 shows a simplified schematic of the waste filter membrane of FIG. 20 incorporated into the cell sieve unit 106P, and excludes other components that can be part of the cell sieve unit 106P. As an example and for simplicity, the schematic shown in FIG. 21 excludes the one or more sequential filters. The cylindrical waste filter is inverted in this design. The cells in solution can come from below and get trapped while the enzyme and washing solution are filtered going up. Given the design of the waste filter membrane 112P, the cell suspension solution (from the tissue digestion chamber) can enter the cell sieve unit and filtration can occur upward by sucking the liquid through the waste port. The design works against gravity and can be operated by suction. The solution goes through the sieves and then is pumped to the lower chamber of the waste filter membrane 112P unit. The liquid passes through the waste filter membrane 112P and then exits via a waste outlet at a top of the cell sieve unit. The waste port can be located at one side. Given the parameters of the waste filter membrane 112P, the solution can pass through the waste filter membrane 112P and the cells can be collected at a bottom portion of the cell sieve unit 106P between the horizontal membrane and the bottom. The collected cells can be inserted into a second device as provided above. It is recognized that other designs of the cell sieve unit 106P (for example, inlet and outlet ports at different locations on the cell sieve unit) can incorporate the horizontal waste filter membrane 112P of FIG. 20.

[00263] Anti-foam structures:

[00264] In the cell isolation devices and methods that include air bubbling, foam development can be observed when the air is introduced into the tissue digestion chamber. Foam may lead to some cell loss since the cells can get trapped in the bubbles. The amount of foam created can depend on at least the following: air flow rate, whether air delivery into the chamber is pulsed or continuous, and the enzyme(s) present in the tissue digestion chamber. FIGS. 22a-22c show foam development during GMP-collagenase I incubation at different flow rates of 50 ml/min, 75 ml/min and 100 ml/min. FIGS. 22a-22c illustrate that foam levels increase as air flow rate increases.

[00265] Anti-foam agents, such as, for example, simethicone emulsion or silicone defoamer (for example, Dupont™ Liveo™ Medical Antifoam C Emulsion), can reduce foam formation. The cell isolation devices of the present application can include one or more anti-foam structures to reduce foam formation in the tissue digestion chamber. FIGS. 23a-23d illustrate examples of anti-foam structures 142, 144, 146 and 148 usable in the cell isolation devices described herein. FIG. 23a shows a plastic insert 150 having a plurality of 2 mm holes 152. FIG. 23b shows a metal insert 154 having a plurality of 3 mm holes 156. FIG. 23c shows a plastic insert 158 having 5 mm holes 160. FIG. 23d shows a metallic insert 162 that is rolled into a spiral structure. In an example, the spiral insert can be comprised of a metal fence-like structure. The structures shown in FIGS. 23a-23d can reduce foam in the tissue digestion chamber by breaking the bubbles up as they are forming. It is recognized that additional designs of the anti-foam structure can be used in the tissue digestion chamber. The plastic or metal inserts or plates can have a plurality of holes, the size of the holes on the plate can vary; in an example, the holes can range from about 1 mm to about 5 mm. The anti-foam structures 142, 144, 146 and 148 can be formed of plastic or metal. The plastics can include those listed above in reference to the cell isolation device or components thereof. Such biocompatible plastics can be cast or injection molded. The metal can include aluminium or any other biocompatible metal.

[00266] FIG. 24 is an expanded view schematic of an exemplary tissue digestion chamber 102Q including a biopsy holder 104Q and anti -foam structure 164. In an example, an elongated structure can be used to secure the skin biopsy. In an example, an air burst filter 166 (which can be similar to the structures in FIGS. 23a and 23b) can be configured at a bottom of the tissue digestion chamber 102Q above the air inlets 168 and 170. Thus, the air burst filter 166 can break up the air as the air is injected into the tissue digestion chamber 102Q. In an example, an anti -foam structure 164 can be configured at a top portion of the tissue digestion chamber 102Q and below a lid of the tissue digestion chamber 102Q. In the example shown in FIG. 24, the anti -foam structure 164 at the top can be a spiral, fence-like structure, similar to that shown in FIG. 23 d.

[00267] FIG. 25 is an expanded view schematic of another exemplary tissue digestion chamber 102R. In the example shown in FIG. 25, the tissue digestion chamber 102R does not include a biopsy holder and the skin biopsy can float freely in the tissue digestion chamber 102R. Similar to the design in FIG. 24, an air burst filter 172 can be used at a bottom of the tissue digestion chamber 102R above the air inlets. In an example, an anti-foam lid 174 (similar to FIGS. 23a or 23b) can be configured at a top portion of the tissue digestion chamber 102R below the lid. The anti -foam lid 174 can have a plurality of holes, and a size of the holes can range between 1-5 mm. The anti -foam hd 174 can be formed of metal or plastic, as provided above in reference to FIGS. 23a-23d.

[00268] It is recognized that the different features in FIGS. 24 and 25 are interchangeable and can be incorporated into the cell isolation devices shown in other FIGS. A cage-like biopsy holder or metal rod, as shown in other FIGS., can be used in the designs of FIGS. 24 and 25. The air burst filter of FIGS. 24 and 25 can be effective, particularly if pulsing is used to inject air into the chamber.

[00269] FIG. 26 is an image of a tissue digestion chamber 102S undergoing testing with the air bubbling system 138 and anti -foam structures 148. As shown in FIG. 26, the tissue digestion chamber 102S includes an air burst filter 176 located between the one or more air bubble inlets and the skin biopsy. The tissue digestion chamber 102S also includes the anti -foam breaker or anti -foam structure 148 above the skin biopsy. In an example, such anti-foam breaker 148 is a plate with a plurality of cylindrical holes. In the example shown in FIG. 26, an additional anti -foam breaker or anti -foam structure 148S can be added onto the tissue digestion chamber 102S - the tissue digestion chamber 102S can be made longer to accommodate the additional anti -foam breaker 148S at the top or an extension piece can be added onto the tissue digestion chamber 102S. Such anti-foam breaker can include a spiral fence-like structure.

[00270] FIGS. 27a-27d are photographic images of foam development with trypsin for different anti-foam structures or breakers. A measuring device is used to aid in comparing the foam levels between the different anti-foam structures. After the trypsin is pumped into the chamber, continuous air flow is delivered into the chamber at 100 ml/min. As shown in FIG. 27d, the metallic spiral insert was most effective at controlling foam development in the chamber. [00271] FIGS. 28a-28d are photographic images of foam development with collagenase for the same anti-foam structures or breakers evaluated in FIGS. 27a-27d. For the evaluations in FIGS. 28a and 28b, continuous air flow was delivered at 75 ml/min. For the evaluations in FIGS. 28c and 28d, air was delivered at 100 ml/min. As shown by the results in FIG. 28d, the metallic spiral insert was also most effective (as compared to the other inserts in FIGS. 28a- 28c) at controlling foam development when collagenase was in the chamber. FIGS. 28a-28c show that there is more foam when collagenase is in the chamber, as compared to trypsin. In both instances (trypsin and collagenase), the spiral insert was most effective at reducing foam/breaking up the bubbles inside the chamber.

[00272] FIG. 29 shows a system 200 that includes devices as described herein but as separate modular units in selectively fluid communication with one another and other components. Thus, the system 200 includes a tissue digestion chamber 202, a cell sieve unit 206 and a filter membrane unit 216. These units can be similar or identical to those described previously. However, the system 200 contemplates the tissue digestion chamber 202, the cell sieve unit 206 and the filter membrane unit 216 can be physically spaced from one another (although coupled in selective fluid communication with one another and with other system components as illustrated in FIG. 29). The system 200 can be housed in a larger device that is not specifically illustrated in FIG. 29. This application contemplates that according to some embodiments the unit(s) described could be combined into two or even one single device as described herein. Furthermore, this application contemplates that the units described could be further sub-divided into further units (four, five, etc.) according to further embodiments.

[00273] Multiple units (e.g., multiple tissue digestion chambers 202, cell sieve units 206, and/or filter membrane units 216 could be utilized in any combination. For example, a single tissue digestion chamber 202 could fluidly connect with a single cell sieve unit 206.

[00274] FIG. 29a shows an embodiment of the system 200 where the single tissue digestion chamber 202 and the single cell sieve unit 206 are fluidly connect with multiple (e.g., two, three, four, five, etc.) filter membrane units 216. This could be fluid connection in parallel (as shown in FIG. 29a) or alternatively the filter membrane units 216 (or other units discussed) could be arranged in series.

[00275] FIG. 29 (and FIG. 29a) shows other components of the system 200 including tissue sample 220, enzyme(s) 222, air 224, wash solution(s) 226 and cell collection 228. It is recognized further components, e.g., tubing, valves, waste collection containers, containers for enzyme(s) are not specifically illustrated in FIG. 29. Additionally, although arrows generally indicate addition of the tissue sample 220, enzyme(s) 222, air 224, wash solution(s) 226 and cell collection 228 to a particular one of the tissue digestion chamber 202, the cell sieve unit 206 and the filter membrane unit 216, it is recognized that the tissue sample 220, enzyme(s) 222, air 224, wash solution(s) 226 and cell collection 228 could be introduced to the system 200 at different ports and/or could be introduced to a different one (or a plurality) of the tissue digestion chamber 202, the cell sieve unit 206 and the filter membrane unit 216 than is specifically shown in FIG. 29. Thus, although air 224 is shown as being provided to the issue digestion chamber 202 in FIG. 29, according to other embodiments, air 224 can be added to the filter membrane unit 216 or the cell sieve unit 206 if desired. The wash solutions(s) 226, shown added to the filter membrane unit 216, could additionally or alternatively be added to the tissue digestion chamber 202. Wash solution(s) 226 can additionally be kept in the cell sieve unit 206, which can act as a reservoir for such solution. Wash solution(s) 226 from the cell sieve unit 206 can be used in the filter membrane unit 216, for example.

[00276] Wash solution(s) 226 such as saline solution(s) can be be flushed through the entire system 200 as a pre-running system check step before the skin tissue biopsy is inserted. The pre-running wash solution 226 can flush and can verify the system’s 200 tightness, lubricate components (e.g., tubes, internal surfaces of units such as the tissue digestion chamber 202, the cell sieve unit 206 and the filter membrane unit 216, and can prevent any debris ends in the system 200.

[00277] Additionally, it is recognized that the system 200 or components thereof could be implemented in a reverse flow arrangement such that inlet ports would become outlet ports and vice versa. Thus, throughout this document the use of the term “inlet” or “outlet” is merely exemplary of the embodiment described. It should be recognized that according to other embodiments an inlet may be an outlet and an outlet may be an inlet. Furthermore, it is recognized that the inlet(s) and outlet(s) described can be utilized with different components of the system. Thus, the same inlet could be used for air and enzyme(s), for example. Thus, an inlet (or outlet) is not limited to a single component of the system. A descriptor such as “air inlet” as used herein merely describes one possible component (air) that the inlet may be receiving. The inlet could also be handling solution (e.g., wash, enzyme(s), etc.). It should be further understood that the number and location of the inlets and/or outlets shown is purely exemplary unless otherwise noted.

Table 3: Process steps with air bubbling and horizontal modular configuration of FIG. 29 or 29a

[00278] The components of the system 200 will now be discussed in regard to specific of the following FIGS. The cell collection 228 can comprise a second device (e.g., a container such as syringe, etc. as discussed herein) for containing single or cell clusters as described herein.

[00279] FIGS. 30, 30a and 30b show the tissue digestion chamber 202. As shown in FIG. 30, the tissue digestion chamber 202 can include a cover 230, outlets 232A, 232B, a sample chamber 234, inlet 236 and outlets 238A and 238B.

[00280] The cover 230 can be removable from a remainder of the tissue digestion chamber 202 to allow the tissue sample to be introduced to the sample chamber 234.

[00281] The embodiment of FIG. 30 does not contemplate the use of biopsy holder(s) or anti-foam structures as described herein. However, one or more of these such as described herein could be utilized according to further examples. The tissue digestion chamber 202 recognizes that anti-foam agents, such as, for example, simethicone emulsion or silicone defoamer (for example, Dupont™ Liveo™ Medical Antifoam C Emulsion), can reduce foam formation to a sufficient degree. These can also coat internal components of the digestion chamber 202, the cell sieve unit 206 and/or the filter membrane unit 216 such that they become siliconized, for example. This can reduce adherence of the cell(s) to the internal components.

[00282] The outlets 232A and 232B can allow air introduced to the tissue digestion chamber 202 (via inlet 236) to escape from the sample chamber 234 and the tissue digestion chamber 202. Enzyme(s) can be introduced to the sample chamber 234 through the inlet 236 along with or prior to the air. Agitation of the tissue sample and enzyme action can cause the tissue sample to break down into cells as previously discussed and illustrated. The cells once obtained from the tissue along with other solution and tissue debris can be transported out of the tissue digestion chamber 202 to the cell sieve unit 206 via the outlets 238 A and 238B. Cell solution after processing through the cell sieve unit 206 and/or the waste filter unit 212 can be in a single cell suspension as further discussed herein.

[00283] FIGS. 30a and 30b show further aspects of the tissue digestion chamber 202 in addition to the inlet 236 and the outlets 238 A and 238B. As best shown in FIG. 30B, the tissue digestion chamber 202 can include an air burst filter 240, a chamber 242, a collection chamber 244, and a wall 246. The air burst filter 240 can include a plurality of apertures 248. The wall 246 can be a radial sidewall of the sample chamber 234 and can include a plurality of ports 250.

[00284] The air burst filter 240 can be similar to those previously discussed having the plurality of apertures 248 therein. The plurality of apertures 248 can cause the air introduced to the tissue digestion chamber 202 to bubble in passing through the air burst filter 240. The bubbles can rise into the sample chamber 234 and agitate the tissue sample therein as described herein.

[00285] The inlet 236 can be positioned adjacent the air burst filter 240 and can communicate air and/or solution (e.g., enzyme, cells, debris etc.) to the chamber 242 beneath the air burst filter 240. This air and/or solution can pass to the sample chamber 234 via the plurality of apertures 248. The chamber 242 can be defined by the air burst filter 240, the wall 246 and an end wall 252 of the tissue digestion chamber 202, for example.

[00286] As shown in FIG. 30B, the air burst filter 240 has a convex shape in cross-section. Similarly, the end wall 252 (sometimes called an end plate) of the tissue digestion chamber 202 can have a convex shape in cross-section. The inlet 236 can be located at an apex 254 of the end wall 252 as shown in FIG.

30B. The convex shape of the air burst filter 240 and the end wall 252 can conduct cells and/or tissue debris away from the inlet 236 and toward and to the collection chamber 244 as further described.

[00287] The collection chamber 244 can be located radially outward of the inlet 236, the air burst filter 240, the chamber 242 and the wall 246. The collection chamber 244 can fluidly communicate with the sample chamber 234 via the plurality of ports 250. The plurality of ports 250 can be radially extending apertures in the wall 246. Some of the of the plurality of ports 250 can fluidly communicate with the chamber 242 beneath the air burst filter 240. The collection chamber 244 can fluidly communicate with the outlets 238 A and 238B to allow the cells and solution to pass from the tissue digestion chamber 202 to the cell sieve unit.

[00288] Once cells are in a suspension, the cells and a solution (e.g., enzyme(s), debris, and/or wash solution) pass through lateral sidewall ports (the plurality of ports 250) in the tissue digestion unit and into the collection chamber 244 positioned outward of sample chamber 234, burst filter 240 and other components of the tissue digestion unit 206.

[00289] FIG. 31 shows another embodiment of a tissue digestion chamber 202A. This tissue digestion chamber 202A differs from the tissue digestion chamber 202 of FIGS. 30-30B in that the end wall 252A of the tissue digestion chamber 202A is concave or tapers downward in shape to the inlet 236 such that the inlet 236 is at a meniscus 256 or other depressed location. A chamber 242A of the tissue digestion chamber 202A may or may not fluidly communicate directly with a collection chamber 244A.

[00290] FIGS. 32-32a show the cell sieve unit 206 according to an example. The cell sieve unit 206 can be constructed in a manner similar or identical to those described previously. The cell sieve unit 206 can include a reservoir 258, a first filter 260, a second filter 262, a main channel 264, inlet 266, ports 268, a sleeve 270 and an outlet 272.

[00291] The reservoir 258 can comprise a central chamber configured to receive the cells and solution (e.g. enzyme(s) and debris) from the tissue digestion chamber 202. This solution can be received via the inlet 266 and can pass along the main channel 264 to be discharged adjacent (within a few mm to a few cm) of the first filter 260. The reservoir 258 in some embodiments can additionally receive via the inlet 266 and main channel 264 wash solution (e.g., up to about 30 ml within an entire volume of the reservoir 258 comprising about 70 ml). However, in other embodiments wash solution is not utilized. In yet further embodiments, wash solution can be utilized within the cell sieve unit 206 to wash internal components prior to adding the suspension (the cells and a solution (e.g., enzyme(s), debris, and/or wash solution)). In yet further embodiments, the wash solution can be utilized to wash internal components within the tissue digestion chamber 202 prior to the introduction of the tissue and/or can be used to wash internal components within the filter membrane unit 216 prior to the introduction of the cells and the solution).

[00292] The construction of the first filter 260 and the second filter 262 has been previously illustrated and discussed in some detail and can comprise a stacked formation as shown. The frame of the first filter 260 and the second filter 262 can be polypropylene. The cells suspended in the solution (e.g., enzymatic digestion solution and/or wash solution) can pass under the influence of gravity (or can be pushed or aspirated using pressurized air or other fluid) through the first filter 260 and the second filter 262 while debris can be retained. The first filter 260 can have a sieve 260A designed to allow cells and other matter of less than 200 pm (or less than 100 pm) to pass therethrough. The second filter 262 has a sieve 262A to allow cells of less than 70 pm to pass therethrough to the outlet 272. The sieve 260A and sieve 262A can be of nylon, for example.

[00293] The sleeve 270 can provide a wall that defines the reservoir 258. The sleeve 270 can be a membrane, bladder or other deformable wall member, for example, that can alter shape to change the size of the reservoir 258. In yet another embodiment the sleeve 270 can be a solid wall structure made of acrylic, polypropylene, or another polymer based component, for example. Air (or another gas or fluid) can be provided to an outer radial side of the sleeve 270 via the ports 268 to regulate pressure within the cell sieve unit 206. In some embodiments it is contemplated internal pressure within the cell sieve unit 206, pumping such as with compressed air or other force (in addition to gravity) could be used to aid in passing the cells through the first filter 260 and/or second filter 262.

[00294] If desired, wash solution if utilized within the reservoir 258 (see discussion above) could be drawn through the outlet 272 and can be provided to other components of the system 200 (FIG. 29) such as the waste filter membrane unit 216 as further described. Additionally, the reservoir 258 can we used to dilute the suspension containing the cells by adding saline or other solution such as from a container through inlet 266 and inlet 264 filling the reservoir 258. [00295] FIGS. 33 and 34 show the waste filter membrane unit 216 according to one embodiment. The waste filter membrane unit 216 can include a lid 274, a housing 276 and a waste filter unit 278. As shown in FIG. 33, the lid 274 can include ports 280A, 280B and 280C. The housing 276 can include ports 282A, 282B and 282C. The waste filter unit 278 can include an inner housing 284, one or more ports 286A and 286B and a waste filter membrane 288.

[00296] FIG. 33 shows an exploded view of the waste filter membrane unit 216. As shown, the waste filter unit 278 can be received within the housing 276. The lid 274 can enclose the waste filter unit 278 within the housing 276. The waste filter unit 278 can be spaced from the housing 276 by a cavity 290. This cavity 290 fluidly communicates with the ports 280A, 280B and 280C and the ports 282A, 282B and 282C.

[00297] The waste filter unit 278 via the inner housing 284 and the waste filter membrane 288 can define a cell concentration chamber 292 (FIG. 34). The waste filter membrane 288 can comprise a sieve and can have a pore size of between 0.4 pm and 5.0 pm. The waste filter membrane can comprise and/or be constructed of any one or combination of: Cellulose Acetate (CA) Membrane, Cellulose Acetate (CA) Membrane, Nitrocellulose (NC) Membrane, Polyethersulfone (PES) Membrane, Nylon (NY) Membrane Polytetrafluoroethylene (PTFE) Membrane, Regenerated Cellulose (RC) Membrane, Polyvinylidene Fluoride (PVDF) Membrane, Polypropylene (PP) Membrane, Polycarbonate Track Etched (PCTE) Membrane, Polyester Track Etched (PETE) Membrane, Polyphenyl sulfone (PPSU), POM-C (Poly acetal (Copolymer)), Polyetheretherketone (PEEK), Polypropylene (PP), mixed cellulose esters (MCE), glass fiber membrane, quarter fiber membrane, silver membrane, aluminium oxide membrane, ceramic membrane, polyacrylonitrile (PAN) membrane or other similar material. The waste filter membrane 288 can be divided by a frame that holds and clamps the porous membrane material (0.4- 5pm pore size) to the inner housing. The frame is represented in the FIG. 33 as single (one “window frame”) but can be a split frame as shown in FIG. 34 (“two window frame”). The cell concentration chamber 292 can fluidly communicate with the one or more ports 286A and/or 286B. The cell concentration chamber 292 collects cells that remain after waste (e.g., enzyme, wash solution or other solution) as further described has passed across the waste filter membrane 288 to the cavity 290. The cells collected in the cell concentration chamber 292 can then be passed through the port 286A to a second device (e.g., another collection device such as a syringe, cell collection unit, cell storage unit, or other device such as an applicator (spray device) as further described herein). Air can pass through the port 286B to maintain desired internal pressure.

[00298] The cavity 290 of the waste filter membrane unit 216 can prefilled with wash solution (e.g., saline solution such as but not limited to Ringer's Lactated, or another solution) using ports 280A, 280B, 280C, and at the same time the cell concentration chamber 292 can be filled since both communicate via the waste filter membrane 288. A cell suspension temporally stored in the cell sieve unit 206 can be injected/pumped through port 272 (FIGS. 32 and 32a) to port 286A. This can cause filling of the cell concentration chamber 292. The cell concentration chamber 292 in different embodiments may have different inner capacities (volumes) such as limited by a displacement body as further discussed and illustrated in FIG. 43. The waste part of cell suspension fill can be forced through waste filter membrane 288 to accommodate the cell concentration chamber 292 inner capacity. This can increase the cell density inside the cell concentration chamber 292 and reduce the available volume within the cell concentration chamber 292 to receive further cell suspension.

[00299] Waste filter membrane 288 will keep cells inside the cell concentration chamber 292. The excess of liquid (waste solution discussed below) of the cell suspension without cells will be mixed with wash solution inside cavity 290 and sent to a waste container (not shown) through ports 282A, 282B, and/or 282C. Injection of fresh wash solution will fill cavity 290 while diluted waste solution will leave the cavity 290 through ports 282A, 282B and/or 282C. Once all cell suspension is filtered, a wash solution from cavity 290 can be used to fill the cell concentration chamber 292 towards cell sieve unit 206 increasing the volume, diluting the cell suspension, and detaching cells from waste filter membrane 288. Diluted cell suspension temporarily stored in from cell sieve unit 206 can be injected/pumped again through port 272 (FIGS. 32 and 32a) to port 286A or 286B, filling the cell concentration chamber 292 to the extent possible with further cell suspension. This process can be repeated several times until the enzyme is diluted sufficiently. Once the dilution process is terminated, the cell suspension (now cells suspended in wash solution - waste solution having exited the waste filter membrane unit 216) can be sent from cell concentration chamber 292 to cell collection (second device) using the port 286A. The above-described process is further illustrated and discussed in regard to FIGS. 35-42.

[00300] FIGS. 35-42 show a process of cell concentration and waste filtering using the waste filter membrane unit 216. As shown in FIGS. 35 and 36 the cavity 290 is filled with wash solution 300 (e.g., Lactated Ringer’s Solution (LRS) or another saline solution) via the ports 280A, 280B and/or 280C. The cell concentration chamber 292 may initially be filled with wash solution as well via the one or more ports 286A.

[00301] A cell suspension 302 (e.g., cells, enzyme and/or wash solution) can be injected into the cell concentration chamber 292 such as via the one of the one or more ports 286A as shown in FIG. 37. The injected solution (e.g., the enzyme and/or wash solution) can pass through the waste filter membrane 288 via diffusion as shown in FIG. 38. However, the cells are too large to pass through the waste filter membrane 288 and are retained in the cell concentration chamber 292. FIG. 38 shows the diffusion of the cell suspension (minus the cells which remain in the cell concentration chamber 292) with the wash solution that passes through the waste filter membrane 288.

[00302] FIG. 39 illustrates a further step of cell concentration and waste filtering of adding additional wash solution 300 to the cavity 290 and/or cell suspension 302 to the cell concentration chamber 292 via the ports 280 A, 280B and/or 280C and/or the one or more ports 286A and/or 286B. Waste solution 304 (e.g., cell suspension of enzyme and/or wash solution minus the cells) can pass from the cavity 290 via the ports 282A, 282B and 282C simultaneously with the filling of the cavity 290 with additional wash solution 300 as shown in FIGS. 39 and 40.

[00303] It should be recognized that the process of FIGS. 39-41 can be repeated multiple times with adding additional wash solution and discharging waste solution to remove waste as desired. Similarly, the process could be repeated with different cell solutions of cells and different ones of the enzymes (e.g., trypsin, gentlyase and collagenase). Wash solution used in FIGS. 39-41 can be provided from the reservoir of the cell sieve unit or can be directed to the reservoir of the cell sieve unit if desired.

[00304] In FIG. 41 a reflux is illustrated where wash solution 300 of FIG. 40 is pumped from cavity 290 through the filter membrane 288 to cell concentration chamber 292 to detach cells from the filter membrane 288. This process can be repeated several times to detach the cells. Then the cell suspension 306 (without enzyme) will be recovered through the port 286A as shown in FIG. 42.

[00305] FIGS. 41 and 42 show the cells concentrated but in the cell suspension 306 such as with the wash solution 300. Wash solution 300 remains in the cavity 290 and any additional volume in the cell concentration chamber 292 that is not occupied by the cells. Collected cells (indicated with C) such as in the cell suspension 306 with the wash solution 300 can be removed from the cell concentration chamber 292 via the port 286A and/or 286B as desired as shown in FIG. 42.

[00306] The process of FIGS. 35-42 can utilize active filtration where the cell suspension is injected into the cell concentration chamber 292 and the cell suspension is forced through the waste filter membrane 288. This process can be reversed where cell suspension is injected into the cell concentration chamber and wash solution can be forced from the cavity 290 into the cell concentration chamber 292. Additionally, one or more rounds of wash solution and/or cell suspension can be injected into the cell concentration chamber 292 and/or cavity 290 (e.g., at steps of FIGS. 39-41) more cells can be become concentrated within the cell concentration chamber 292 and/or the cells can be removed from the filter membrane 288 such as using the reflux of FIG. 41. Wash solution injected into the cavity 290 as shown in FIG. 41 can pass through the waste filter membrane 288 and can detach cells trapped on the walls of the waste filter membrane 288. Additionally, in some embodiments reflux or flushing back could be performed using air (e.g., compressed air) to detach cells and/or to further concentrate cells in the cell concentration chamber 292. Airflow rate could be 50-300 mL/min. Flushing back air (or with another solution) could also be performed on additional units such as at a system level. Thus, not only would the waste filter membrane unit 212 be flushed but also with the tissue digestion chamber 202 and/or the cell sieve unit 206 can be flushed.

[00307] FIG. 43 shows an alternative embodiment of a waste filter membrane unit 216A. The waste filter membrane unit 216A can have a similar design as the waste filter membrane unit 216 described herein but can differ in that the cell concentration chamber 292 contains a displacement body 294A. The displacement body 294A can be cylindrically shaped and can have conically shaped first and/or second ends, for example. The displacement body 294A can increase the concentration of cells obtained from the cell concentration chamber 292. In particular, the displacement body 294A reduces the volume of the waste filter membrane unit 216A (e.g., from about 12 mL to 4 mL according to one example. This can increase the concentration of cells from about 0.5 M cells/mL to about 2.5M cells/mL during the filtering using the process described above. One is able to control the inner capacity of the cell concentration chamber 292 depending on the size of the displacement body 294A. Additionally, wash solution can be directed by the displacement body 294A shape against the waste filter membrane 288 to detach the cells trapped against the waste filter membrane 288. The cell suspension (substantially free of enzyme) can be recovered from the cell concentration chamber 292 using the port(s) 286A and/or 286B described previously. The cells can be at a desired volume and a higher concentration as compared to the prior embodiment of FIGS. 33-42.

[00308] FIGS. 44 and 44a show another alternative embodiment of a waste filter membrane unit 216B. The waste filter membrane unit 216B can have a similar design as the waste filter membrane unit 216 described herein but can differ in that the waste filter membrane 216B can include one or more spacers 296B that force wash solution or compressed air (bubbles) entering cavity 290 against the waste filter membrane 288 to detach the cells trapped against the waste filter membrane 288 within the cell concentration chamber 292 back to settle freely within the cell concentration chamber 292. The ports 280A and/or 280B and/or 282A, 282B could be selectively opened and closed to direct wash fluid against the one or more spacers 296B and against the waste filter membrane 288. [00309] Clinical Use:

[00310] Cell-spray autografting is an innovative early treatment option for deep partial-thickness burn wounds. As an alternative to non-operative management, cell-spray autografting can achieve rapid wound re- epithelialization, particularly in large wounds. When compared to traditional mesh autografting for deep partial-thickness burn wounds, cell-spray autografting can accomplish re-epithelialization with a much smaller donor site. [00311] The cell isolation device as disclosed herein can be used to isolate cells from other tissues by changing the initial tissue sources and using the proper enzymes. We envision cell isolation from tissues like liver, adipose tissue amongst others to address clinical therapeutic options to treat liver disease or diabetic ulcers as an example. Below is a tabular view of a protocol, providing a summary of the steps for when a skin biopsy is used and followed by use of a cell spray gun to administer isolated single cell suspensions of cells to a subject in need thereof.

[00312] Tabular view of an Embodiment of a System Protocol:

[00313] Step 9 (“SkinGun Spray Process) above refers to the cell spray gun (cell spray device) and methods described in U.S. Patent No. 9,505,000, U.S. Patent No. 9,610,430, U.S. Pat. No. 10,376,658, PCT Patent Application No. PCT/US2017/037274, PCT Patent Application No. PCT/EP2017064094, and German Patent No. DE102011100450B4, as well as variations thereof.

[00314] A device for isolating cells can include a tissue digestion chamber configured to receive enzymes and biopsy tissue to cause release of cells from the biopsy tissue. The device can include a filter unit comprising one or more filters to separate tissue debris from the cells. The device can include a waste outlet and a cell outlet for releasing filtered cells from the device.

[00315] The device can be modular such that specific components of the device can be customized or selected by the user for a particular application. [00316] The one or more filters can include three sequential filters of decreasing pore size.

[00317] The device can include a waste filter membrane in proximity to the waste outlet. The waste filter membrane can have a membrane pore size that is smaller than the smallest pore size of the one or more filters.

[00318] The waste filter membrane can be located in the tissue digestion chamber.

[00319] The waste filter membrane can be located in the cell sieve unit in proximity to a syringe port.

[00320] The waste filter membrane can include a radially oriented membrane filter.

[00321] The waste filter membrane can include a horizontal filter.

[00322] The device can include multiple inlets for at least one of enzymes, air and washing solutions. The device can include multiple outlets for at least one of enzymes, air and washing solutions. The multiple inlets can include two inlets for air to enable an air bubbling effect in the tissue digestion chamber. The multiple outlets can include more than one air outlet, including four air outlets.

[00323] The tissue digestion chamber can include one or more biopsy holders for retaining the biopsy tissue during tissue digestion.

[00324] The biopsy holder can include a cage-like structure insertable into the tissue digestion chamber and having open and closed positions. The cage-like structure can include an inner cylinder and an outer cylinder. The cage-like structure can have holes of different sizes. The cage-like structure can have a different shape on one end or additional structures to reduce the foam formation during the tissue digestion or cell liberation.

[00325] The biopsy holder can include one or more snap-on supports to secure the biopsy holder in the closed position. The one or more snap-on supports can include four snap-on supports along a longitudinal length of the biopsy holder.

[00326] The biopsy holder can include a metal rod insertable into the tissue digestion chamber and extending along a longitudinal length of the tissue digestion chamber. The biopsy holder can include clips that attach to the metal rod for retaining the biopsy tissue.

[00327] The tissue digestion chamber can include one or more anti-foam structures. The anti-foam structures can include plastic or metallic inserts or plates with a plurality of holes. The anti -foam structures can include a spiral insert with a fence-like structure. The tissue digestion chamber can include one or more air burst filters between the air inlets and the skin biopsy.

[00328] The enzymes received in the tissue digestion chamber can include one or more of trypsin, gentlyase, dispase and collagenase.

[00329] The isolated cells can be used in in vivo, ex vivo, and in vitro applications.

[00330] The biopsy tissue can be selected from skin, liver, bone and other tissues and organs.

[00331] A method of isolating cells can include providing a cell isolation device, the cell isolation device comprising a tissue digestion chamber, a filter unit, a waste outlet and a cell outlet. The method can include containing the skin biopsy in or on a biopsy holder, inserting the biopsy holder into the tissue digestion chamber, and digesting the skin biopsy using one or more enzymes. The method can include passing a solution containing the isolated cells into the filter unit, the filter unit containing one or more filters to separate tissue debris and cell aggregates from single cells. The method can include passing the solution containing the single cells into a waste filter membrane to retain the cells and collecting the cells for delivery to a syringe. The waste filter membrane can be located in the tissue digestion chamber or the filter unit. [00332] The step of digesting the skin biopsy can include injecting air into the tissue digestion chamber and producing air bubbles to move the skin biopsy as it is being digested by the one or more enzymes to agitate the tissue.

[00333] The step of digesting the skin biopsy can include using anti-foam structures to reduce foaming inside the tissue digestion chamber during air bubbling.

[00334] The step of digesting the skin biopsy can include shaking the device to agitate the tissue.

[00335] The above detailed description includes references to the accompanying drawings, which form a part of the detailed description. The drawings show, by way of illustration, specific embodiments in which the invention can be practiced. These embodiments are also referred to herein as “examples.” Such examples can include elements in addition to those shown or described. However, the present inventors also contemplate examples in which only those elements shown or described are provided. Moreover, the present inventors also contemplate examples using any combination or permutation of those elements shown or described (or one or more aspects thereof), either with respect to a particular example (or one or more aspects thereof), or with respect to other examples (or one or more aspects thereof) shown or described herein. [00336] The above description is intended to be illustrative, and not restrictive. For example, the above-described examples (or one or more aspects thereof) may be used in combination with each other. Other embodiments can be used, such as by one of ordinary skill in the art upon reviewing the above description. The Abstract is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Also, in the above Detailed Description, various features may be grouped together to streamline the disclosure. This should not be interpreted as intending that an unclaimed disclosed feature is essential to any claim. Rather, inventive subject matter may lie in less than all features of a particular disclosed embodiment.

Thus, the following claims are hereby incorporated into the Detailed Description as examples or embodiments, with each claim standing on its own as a separate embodiment, and it is contemplated that such embodiments can be combined with each other in various combinations or permutations. The scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

[00337] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. [00338] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification.

[00339] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention.

[00340] Bibliography — the disclosures of which are hereby incorporated by reference in their entirety.

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[00342] 2. Esteban- Vives R, Choi MS, Young M, Over P, Ziembicki J, Corcos A, Gerlach JC: Second-degree burn injuries with six etiologies treated with autologous noncultured cell-spray grafting. Burns 2016, 42(7) :e99-e 106 [00343] 3. Gerlach JC, Johnen C, Ottoman C, Brautigam K, Plettig J, Belfekroun C, Munch S, Hartmann B: Method for autologous single skin cell isolation for regenerative cell spray transplantation with non-cultured cells. The International journal of artificial organs 2011, 34(3):271

[00344] 4. Ollier L: Sue les greffes cutanees ou autopl astiques. Bull Acad Med Paris 1872, 41(3): 1 :234

[00345] 5. Reverdin J: THE CLASSIC REPRINT GREFFE EPIDERMIQUE-EXPERIENCE FAITE DANS LE SERVICE DE M. LE DOCTEUR GUYON, A LHOPITAL NECKER. Plast Reconstr Surg 1968, 41(1):79-81 [00346] 6. Wolfe JR: A new method of performing plastic operations. British medical journal 1875, 2(768):360

[00347] 7. Blandford Jr S, Garcia F: Case report: successful homogenous skin graft in a severe burn using an identical twin as donor. Plast Reconstr Surg 1953, 1 l(l):31-35

[00348] 8. Tanner JC, Jr., Shea PC, Jr., Bradley WH, Vandeput JJ: Large- mesh skin grafts. Plast Reconstr Surg 1969, 44(5):504-506

[00349] 9. Tanner JC, Jr., Vandeput J, Olley JF: THE MESH SKIN GRAFT. Plast Reconstr Surg 1964, 34:287-292

[00350] 10. Meek C: Extensive severe burn treated with enzymatic debridement and microdermagrafting: case report. The American surgeon 1963, 29:61

[00351] 11. Kreis R, Mackie D, Hermans R, Vloemans A: Expansion techniques for skin grafts: comparison between mesh and Meek island (sandwich-) grafts. Burns 1994, 20:S39-S42

[00352] 12. Johnen C, Hartmann B, Steffen I, Brautigam K, Witascheck T, Toman N, Kuntscher MV, Gerlach JC: Skin cell isolation and expansion for cell transplantation is limited in patients using tobacco, alcohol, or are exhibiting diabetes mellitus. Burns 2006, 32(2): 194-200

[00353] 13. Hartmann B, Ekkernkamp A, Johnen C, Gerlach JC, Belfekroun C, Kontscher MV: Sprayed Cultured Epithelial Autografts for Deep Dermal Burns of the Face and Neck. Annals of Plastic Surgery 2007, 58(l):70-73 [00354] 14. Gerlach JC, Johnen C, McCoy E, Brautigam K, Plettig J, Corcos A: Autologous skin cell spray -transplantation for a deep dermal burn patient in an ambulant treatment room setting. Burns 2011, 37(4):el9-e23

[00355] 15. Esteban- Vives R, Corcos A, Choi MS, Young MT, Over P, Ziembicki J, Gerlach JC: Cell-spray auto-grafting technology for deep partialthickness burns: Problems and solutions during clinical implementation. Burns 2018, 44(3):549-559.

[00356] 16. Esteban- Vives R, Young MT, Zhu T, Beiriger J, Pekor C, Ziembicki J, Corcos A, Rubin P, Gerlach JC: Calculations for reproducible autologous skin cell spray grafting. Burns 2016, 42(8): 1756-1765.

[00357] Claim Related Examples [00358] In some aspects, the examples described herein relate to a device for isolating cells from biopsy tissue, the device including: a tissue digestion unit configured to receive biopsy tissue, one or more enzymes, and one or more washing solutions, the one or more enzymes causing release of cells from the biopsy tissue; a filter unit in fluid connection with the tissue digestion unit, the filter unit including one or more sieves to separate tissue debris from the cells; a cell outlet to release filtered cells from the device; and a filter membrane configured for passing a waste solution out of the device.

[00359] In some aspects, the examples described herein relate to a device, wherein the filter membrane is located between the one or more sieves and the cell outlet, and the filter membrane is configured for collecting the cells.

[00360] In some aspects, the examples described herein relate to a device wherein the one or more sieves include three sequential sieves of decreasing pore size.

[00361] In some aspects, the examples described herein relate to a device wherein the pore sizes of the three sequential sieves are between about 70 and about 200 pm.

[00362] In some aspects, the examples described herein relate to a device wherein a pore size of the filter membrane is less than a smallest pore size of the one or more sieves.

[00363] In some aspects, the examples described herein relate to a device wherein the filter membrane is located in the filter unit.

[00364] In some aspects, the examples described herein relate to a device wherein the filter membrane is a cylinder having an open top and an open bottom, and a membrane material forms at least a portion of an exterior of the cylinder

[00365] In some aspects, the examples described herein relate to a device wherein a pore size of the membrane material is between about 0.4 and about 5 pm.

[00366] In some aspects, the examples described herein relate to a device wherein the filter membrane is located in the tissue digestion unit. [00367] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit includes a biopsy holder for retaining the biopsy tissue during tissue digestion.

[00368] In some aspects, the examples described herein relate to a device wherein the biopsy holder includes an outer cylinder having openings, the outer cylinder movable between open and closed positions for receiving the biopsy tissue inside the outer cylinder, and wherein the outer cylinder is insertable into the tissue digestion chamber and the one or more enzymes access the biopsy tissue through the openings in the outer cylinder.

[00369] In some aspects, the examples described herein relate to a device wherein the biopsy holder includes an inner cylinder that the skin biopsy is rolled onto, and the inner cylinder is inserted into the outer cylinder.

[00370] In some aspects, the examples described herein relate to a device wherein the biopsy holder includes a metal rod insertable into the tissue digestion chamber to extend along a length of the tissue digestion chamber. [00371] In some aspects, the examples described herein relate to a device wherein the biopsy holder includes one or more clips attachable to the metal rod to retain the biopsy tissue.

[00372] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit includes an anti-form structure configured to break up bubbles formed in the tissue digestion unit when air is pumped into the tissue digestion unit.

[00373] In some aspects, the examples described herein relate to a device wherein the anti -foam structure is a circular plate having a plurality of holes formed in the plate.

[00374] In some aspects, the examples described herein relate to a device wherein the anti-foam structure is a rolled-up metal structure having a spiral cross section.

[00375] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit includes an air burst filter near an air inlet of the tissue digestion unit, the air burst filter including a plurality of holes formed in a plate. [00376] In some aspects, the examples described herein relate to a device wherein the device is one piece and a valve controls flow of fluid from the tissue digestion unit to the filter unit.

[00377] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit and the filter unit are separate from one another and attached via a connector.

[00378] In some aspects, the examples described herein relate to a device wherein the tissue digestion chamber includes: multiple inlets for receiving at least one of enzymes, air and washing solutions into the tissue digestion chamber; and multiple outlets for releasing at least one of enzymes, air and washing solutions from the tissue digestion chamber.

[00379] In some aspects, the examples described herein relate to a device further including: a syringe port located in proximity to the filter membrane and configured to receive a syringe for collecting the cells from the filter membrane. [00380] In some aspects, the examples described herein relate to a device for isolating cells, the device including: a tissue digestion chamber including: air inlets for pumping air into the tissue digestion chamber; solution inlets for injecting enzymes and washing solutions into the tissue digestion chamber; and air outlets for releasing air out of the tissue digestion chamber, the tissue digestion chamber configured to receive biopsy tissue and one or more enzymes, the one or more enzymes causing release of cells from the biopsy tissue; a cell filter unit fluidly connected to the tissue digestion chamber to receive from the tissue digestion chamber a solution containing cells, the cell filter unit including one or more sieves to separate tissue debris from the cells; a filter membrane for passing the solution through the filter membrane and collecting the cells; a waste outlet configured to receive the solution from the filter membrane; and a syringe outlet in proximity to the filter membrane and configured for connection to a syringe to extract the collected cells from the device and into the syringe.

[00381] In some aspects, the examples described herein relate to a device wherein the tissue digestion chamber further includes a biopsy holder for retaining the biopsy tissue.

[00382] In some aspects, the examples described herein relate to a device further including one or more anti-foam structures in or near the tissue digestion chamber, the one or more anti-foam structures configured to break up bubbles formed in the tissue digestion chamber created by air pumped into the tissue digestion chamber.

[00383] In some aspects, the examples described herein relate to a device wherein the one or more anti-foam structures includes a circular plate having a plurality of holes formed in the plate.

[00384] In some aspects, the examples described herein relate to a device wherein the one or more anti-foam structures includes a rolled-up structure having a spiral cross section.

[00385] In some aspects, the examples described herein relate to a device wherein the one or more sieves include three sequential sieves of decreasing pore size.

[00386] In some aspects, the examples described herein relate to a device wherein the filter membrane is a cylinder having an open top and an open bottom, and at least a portion of the cylinder is formed by a membrane material having a pore size between about 0.4 and about 5 pm.

[00387] In some aspects, the examples described herein relate to a method of isolating cells from biopsy tissue, the method including: providing a cell isolation device including a tissue digestion chamber, a filter unit and a cell outlet; inserting biopsy tissue into the tissue digestion chamber; inserting one or more enzymes into the tissue digestion chamber; digesting the biopsy tissue into cells using the one or more enzymes; passing the one or more enzymes out of the tissue digestion chamber; inserting a washing solution into the tissue digestion chamber to wash the biopsy tissue and released cells; filtering a solution containing the cells through one or more sieves in the filter unit to separate tissue debris and cell aggregates from single cells; passing the solution containing the single cells through a waste filter membrane to retain the cells and remove the solution from the device; and collecting the cells for delivery to a syringe via the cell outlet.

[00388] In some aspects, the examples described herein relate to a method, wherein inserting biopsy tissue into the tissue digestion chamber includes: retaining biopsy tissue in or on a biopsy holder; and inserting the biopsy holder and the biopsy tissue into the tissue digestion chamber. [00389] In some aspects, the examples described herein relate to a method wherein digesting the biopsy tissue includes injecting air into the tissue digestion chamber and producing air bubbles to agitate the biopsy tissue as it is being digested by the one or more enzymes.

[00390] In some aspects, the examples described herein relate to a method wherein the air is injected into the tissue digestion chamber via two air inlets of the tissue digestion chamber.

[00391] In some aspects, the examples described herein relate to a method wherein the air is injected continuously at a flow rate between about 50 and about 300 ml/min.

[00392] In some aspects, the examples described herein relate to a method wherein the air is injected via a pulse at a constant or variable airflow between about 50 and about 300 ml/min.

[00393] In some aspects, the examples described herein relate to a method wherein digesting the biopsy tissue includes shaking the device to agitate the biopsy tissue as it is being digested by the one or more enzymes.

[00394] In some aspects, the examples described herein relate to a method further including: controlling foaming in the tissue digestion chamber using one or more anti-foam structures.

[00395] In some aspects, the examples described herein relate to a method wherein passing the solution containing the cells into the filter unit includes regulating the flow of the solution into the filter unit with a valve.

[00396] In some aspects, the examples described herein relate to a method wherein inserting one or more enzymes into the tissue digestion chamber includes inserting at least one of trypsin, dispase, collagenase and gentlyase. [00397] In some aspects, the examples described herein relate to a method, wherein inserting one or more enzymes includes inserting two or more enzymes and each enzyme is inserted sequentially, and the following steps are performed for each enzyme: passing the one or more enzymes out of the tissue digestion chamber; and inserting a washing solution into the tissue digestion chamber to wash the biopsy tissue and released cells. [00398] In some aspects, the examples described herein relate to a method wherein the biopsy tissue is selected from skin, liver, bone and other tissues and organs.

[00399] In some aspects, the examples described herein relate to a method wherein the cells collected in the syringe are used in in vivo, ex vivo, and in vitro applications.

[00400] In some aspects, the examples described herein relate to a device for isolating cells from biopsy tissue, the device including: a tissue digestion unit configured to receive biopsy tissue, and one or more enzymes, the one or more enzymes causing release of cells from the biopsy tissue; a cell sieve unit in fluid connection with the tissue digestion unit, the cell sieve unit including one or more sieves to separate tissue debris from the cells; a cell outlet to release the cells after the cells are separated from the tissue debris; and a filter membrane configured for passing a waste solution that includes the tissue debris and the one or more enzymes.

[00401] In some aspects, the examples described herein relate to a device wherein the one or more sieves include two sequential sieves of decreasing pore size.

[00402] In some aspects, the examples described herein relate to a device wherein the decreasing pore sizes of the two sequential sieves are between about 70 and about 200 pm.

[00403] In some aspects, the examples described herein relate to a device wherein a pore size of the filter membrane is less than a smallest pore size of the one or more sieves.

[00404] In some aspects, the examples described herein relate to a device wherein the filter membrane is part of a waste filter membrane unit separate from the tissue digestion unit and the cell sieve unit and is in fluid communication with the cell sieve unit.

[00405] In some aspects, the examples described herein relate to a device wherein the waste filter membrane unit has a cell concentration chamber to retain the cells, wherein the filter membrane forms one or more walls of the cell concentration chamber. [00406] In some aspects, the examples described herein relate to a device wherein the cell concentration chamber is in fluid communicate with a second chamber via the filter membrane, wherein the second chamber is configured to receive a wash solution and the filter membrane is configured to pass the wash solution into the cell concentration chamber to form a suspension with the cells therein.

[00407] In some aspects, the examples described herein relate to a device wherein the cell concentration chamber has at least one inlet to receive the wash solution in combination with a second inlet to the second chamber.

[00408] In some aspects, the examples described herein relate to a device wherein the filter membrane has a pore size of between about 0.4 and about 5 pm.

[00409] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit includes a biopsy holder for retaining the biopsy tissue during tissue digestion.

[00410] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit includes an air burst filter near an air inlet of the tissue digestion unit, the air burst filter including a plurality of holes formed in a plate.

[00411] In some aspects, the examples described herein relate to a device wherein the air burst filter is configured to create bubbles from air passing through the air burst filter.

[00412] In some aspects, the examples described herein relate to a device wherein once the cells are in a suspension, the cells and one or more of the waste solution or a wash solution can pass through lateral sidewall ports in the tissue digestion unit and into a collection chamber.

[00413] In some aspects, the examples described herein relate to a device wherein a base of the collection chamber communicates with one or more outlets of the tissue digestion unit.

[00414] In some aspects, the examples described herein relate to a device wherein the plate of the air burst filter has a concave shape and the plurality of holes pass through the plate. [00415] In some aspects, the examples described herein relate to a device wherein an end of the tissue digestion unit is spaced from the air burst filter, and wherein the end of the tissue digestion unit has one of a concave shape or convex shape with the air inlet being at one of an apex or meniscus thereof. [00416] In some aspects, the examples described herein relate to a device wherein the end of the tissue digestion unit and the air burst filter form a cavity having ports to direct cells that pass from the tissue digestion unit through the air burst filter to one or more outlets of the tissue digestion unit.

[00417] In some aspects, the examples described herein relate to a device wherein the tissue digestion unit includes: multiple inlets for receiving at least one of enzyme, air and washing solution into the tissue digestion unit; and multiple outlets for releasing at least one of enzyme, air and washing solution from the tissue digestion unit.

[00418] In some aspects, the examples described herein relate to a device further including: a port located in proximity to the filter membrane and configured to receive a syringe for collecting the cells from the filter membrane. [00419] In some aspects, the examples described herein relate to a device wherein when the cells are isolated the cells are in a single cell suspension. [00420] In some aspects, the examples described herein relate to a device for isolating cells, the device including: a tissue digestion chamber including: one or more air inlets for pumping air into the tissue digestion chamber; one or more solution inlets for injecting enzyme and washing solution into the tissue digestion chamber; and one or more air outlets for releasing air out of the tissue digestion chamber, the tissue digestion chamber configured to receive biopsy tissue and enzyme, the enzyme causing release of cells from the biopsy tissue; a cell sieve unit fluidly connected to the tissue digestion chamber to receive from the tissue digestion chamber a solution containing cells and tissue debris, the cell sieve unit including one or more sieves to separate the tissue debris from the cells; a filter membrane configured to allow the enzyme and washing solution to pass therethrough, the filter membrane retaining the cells; a waste outlet configured to receive the enzyme and washing solution from the filter membrane; and an outlet in proximity to the filter membrane and configured for connection to a second device to extract the cells retained by the filter membrane.

[00421] In some aspects, the examples described herein relate to a device wherein the tissue digestion chamber further includes a biopsy holder for retaining the biopsy tissue.

[00422] In some aspects, the examples described herein relate to a device wherein the one or more sieves include two sequential sieves of decreasing pore size.

[00423] In some aspects, the examples described herein relate to a device wherein the filter membrane is formed by a membrane material having a pore size between about 0.4 and about 5 pm.

[00424] In some aspects, the examples described herein relate to a device wherein the filter membrane is part of a waste filter membrane unit separate from the tissue digestion chamber and the cell sieve unit and is in fluid communication with the cell sieve unit.

[00425] In some aspects, the examples described herein relate to a device wherein the waste filter membrane unit has a cell concentration chamber to retain the cells, wherein the filter membrane forms one or more walls of the cell concentration chamber, and wherein the cell concentration chamber is in fluid communicate with a second chamber via the filter membrane, wherein the second chamber is configured to receive a wash solution and the filter membrane is configured to pass the wash solution into the cell concentration chamber to form a suspension with the cells therein.

[00426] In some aspects, the examples described herein relate to a device wherein the tissue digestion chamber has an air burst filter near an air inlet of the tissue digestion chamber, the air burst filter including a plurality of holes formed in a plate, wherein the air burst filter is configured to create bubbles from air passing through the plurality of holes of the air burst filter.

[00427] In some aspects, the examples described herein relate to a method of isolating cells from biopsy tissue, the method including: inserting the biopsy tissue into a tissue digestion chamber; inserting one or more enzymes into the tissue digestion chamber; digesting the biopsy tissue to release the cells using the one or more enzymes; filtering a solution containing the cells through one or more sieves to separate tissue debris from the cells; passing the solution containing the cells through a waste filter membrane to retain the cells; and collecting the cells for delivery to a second device.

[00428] In some aspects, the examples described herein relate to a method further including inserting a washing solution into the tissue digestion chamber to wash the biopsy tissue and the cells.

[00429] In some aspects, the examples described herein relate to a method further including: passing the solution containing the cells into a cell concentration chamber; diluting the solution containing the cells within the cell concentration chamber; and passing the solution once diluted through the waste filter membrane to retain the cells within the cell concentration chamber but allow the solution to pass to or from the cell concentration chamber.

[00430] In some aspects, the examples described herein relate to a method wherein diluting the solution containing the cells within the cell concentration chamber includes pumping a wash solution into a second chamber that fluidly communicates with the cell concentration chamber through the waste filter membrane.

[00431] In some aspects, the examples described herein relate to a method wherein the pumping the wash solution includes pumping the wash solution into the cell concentration chamber.

[00432] In some aspects, the examples described herein relate to a method wherein the solution without the cells diffuses across the waste filter membrane.

[00433] In some aspects, the examples described herein relate to a method wherein the wash solution diffuses across the waste filter membrane and into the cell concentration chamber to form a suspension with the cells.

[00434] In some aspects, the examples described herein relate to a method further including repeating pumping the wash solution into the second chamber that fluidly communicates with the cell concentration chamber through the waste filter membrane and further diffusing the wash solution across the waste filter membrane.

[00435] In some aspects, the examples described herein relate to a method wherein at least a portion of the wash solution is from a cell sieve unit that performs the filtering the solution containing the cells through one or more sieves.

[00436] In some aspects, the examples described herein relate to a method wherein inserting biopsy tissue into the tissue digestion chamber includes: retaining the biopsy tissue in or on a biopsy holder; and inserting the biopsy holder and the biopsy tissue into the tissue digestion chamber.

[00437] In some aspects, the examples described herein relate to a method wherein digesting the biopsy tissue includes injecting air into the tissue digestion chamber and producing air bubbles to agitate the biopsy tissue as it is being digested by the one or more enzymes.

[00438] In some aspects, the examples described herein relate to a method wherein the air is injected into the tissue digestion chamber continuously at a flow rate between about 50 and about 300 ml/min.

[00439] In some aspects, the examples described herein relate to a method wherein the air is injected via a pulse at a constant or variable airflow between about 50 and about 300 ml/min.

[00440] In some aspects, the examples described herein relate to a method wherein digesting the biopsy tissue includes shaking the tissue digestion chamber to agitate the biopsy tissue as it is being digested by the one or more enzymes.

[00441] In some aspects, the examples described herein relate to a method further including: controlling foaming in the tissue digestion chamber using one or more anti-foam agents.

[00442] In some aspects, the examples described herein relate to a method further including siliconizing components including the tissue digestion chamber and the waste filter membrane with the one or more anti-foam agents.

[00443] In some aspects, the examples described herein relate to a method wherein inserting one or more enzymes into the tissue digestion chamber includes inserting at least one of trypsin, dispase, collagenase and gentlyase. [00444] In some aspects, the examples described herein relate to a method wherein inserting one or more enzymes includes inserting two or more enzymes and each enzyme is inserted sequentially, and performing for each enzyme: passing one of the one or more the enzymes out of the tissue digestion chamber; and inserting a washing solution into the tissue digestion chamber to wash the biopsy tissue and released cells.

[00445] In some aspects, the examples described herein relate to a method wherein the biopsy tissue is selected from skin, liver, umbilical cord, bone and other tissues and organs.

[00446] In some aspects, the examples described herein relate to a method wherein the cells collected in the second device are in a single cell suspension and are used in in vivo, ex vivo, and in vitro applications.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A device for isolating cells from biopsy tissue, the device comprising: a tissue digestion unit configured to receive biopsy tissue, and one or more enzymes, the one or more enzymes causing release of cells from the biopsy tissue; a cell sieve unit in fluid connection with the tissue digestion unit, the cell sieve unit comprising one or more sieves to separate tissue debris from the cells; a cell outlet to release the cells after the cells are separated from the tissue debris; and a filter membrane configured for passing a waste solution that includes the tissue debris and the one or more enzymes.

2. The device of claim 1 wherein the one or more sieves comprise two sequential sieves of decreasing pore size.

3. The device of claim 2 wherein the decreasing pore sizes of the two sequential sieves are between about 70 and about 200 pm.

4. The device of claim 2 wherein a pore size of the filter membrane is less than a smallest pore size of the one or more sieves.

5. The device of claim 1 wherein the filter membrane is part of a waste filter membrane unit separate from the tissue digestion unit and the cell sieve unit and is in fluid communication with the cell sieve unit.

6. The device of claim 5 wherein the waste filter membrane unit has a cell concentration chamber to retain the cells, wherein the filter membrane forms one or more walls of the cell concentration chamber.

7. The device of claim 6 wherein the cell concentration chamber is in fluid communicate with a second chamber via the filter membrane, wherein the

78 second chamber is configured to receive a wash solution and the filter membrane is configured to pass the wash solution into the cell concentration chamber to form a suspension with the cells therein.

8. The device of claim 7 wherein the cell concentration chamber has at least one inlet to receive the wash solution in combination with a second inlet to the second chamber.

9. The device of claim 1 wherein the filter membrane has a pore size of between about 0.4 and about 5 pm.

10. The device of claim 1 wherein the tissue digestion unit comprises a biopsy holder for retaining the biopsy tissue during tissue digestion.

11. The device of claim 1 wherein the tissue digestion unit comprises an air burst filter near an air inlet of the tissue digestion unit, the air burst filter including a plurality of holes formed in a plate.

12. The device of claim 11 wherein the air burst filter is configured to create bubbles from air passing through the air burst filter.

13. The device of claim 12 wherein once the cells are in a suspension, the cells and one or more of the waste solution or a wash solution can pass through lateral sidewall ports in the tissue digestion unit and into a collection chamber.

14. The device of claim 13 wherein a base of the collection chamber communicates with one or more outlets of the tissue digestion unit.

15. The device of claim 11 wherein the plate of the air burst filter has a concave shape and the plurality of holes pass through the plate.

16. The device of claim 15 wherein an end of the tissue digestion unit is spaced from the air burst filter, and wherein the end of the tissue digestion unit

79 has one of a concave shape or convex shape with the air inlet being at one of an apex or meniscus thereof.

17. The device of claim 16 wherein the end of the tissue digestion unit and the air burst filter form a cavity having ports to direct cells that pass from the tissue digestion unit through the air burst filter to one or more outlets of the tissue digestion unit.

18. The device of claim 1 wherein the tissue digestion unit comprises: multiple inlets for receiving at least one of enzyme, air and washing solution into the tissue digestion unit; and multiple outlets for releasing at least one of enzyme, air and washing solution from the tissue digestion unit.

19. The device of claim 1 further comprising: a port located in proximity to the filter membrane and configured to receive a syringe for collecting the cells from the filter membrane.

20. The device of claim 1 wherein when the cells are isolated the cells are in a single cell suspension.

21. A device for isolating cells, the device comprising: a tissue digestion chamber comprising: one or more air inlets for pumping air into the tissue digestion chamber; one or more solution inlets for injecting enzyme and washing solution into the tissue digestion chamber; and one or more air outlets for releasing air out of the tissue digestion chamber, the tissue digestion chamber configured to receive biopsy tissue and enzyme, the enzyme causing release of cells from the biopsy tissue; a cell sieve unit fluidly connected to the tissue digestion chamber to receive from the tissue digestion chamber a solution containing cells and tissue

80 debris, the cell sieve unit comprising one or more sieves to separate the tissue debris from the cells; a filter membrane configured to allow the enzyme and washing solution to pass therethrough, the filter membrane retaining the cells; a waste outlet configured to receive the enzyme and washing solution from the filter membrane; and an outlet in proximity to the filter membrane and configured for connection to a second device to extract the cells retained by the filter membrane.

22. The device of claim 21 wherein the tissue digestion chamber further comprises a biopsy holder for retaining the biopsy tissue.

23. The device of claim 21 wherein the one or more sieves include two sequential sieves of decreasing pore size.

24. The device of claim 21 wherein the filter membrane is formed by a membrane material having a pore size between about 0.4 and about 5 pm.

25. The device of claim 21 wherein the filter membrane is part of a waste filter membrane unit separate from the tissue digestion chamber and the cell sieve unit and is in fluid communication with the cell sieve unit.

26. The device of claim 25 wherein the waste filter membrane unit has a cell concentration chamber to retain the cells, wherein the filter membrane forms one or more walls of the cell concentration chamber, and wherein the cell concentration chamber is in fluid communicate with a second chamber via the filter membrane, wherein the second chamber is configured to receive a wash solution and the filter membrane is configured to pass the wash solution into the cell concentration chamber to form a suspension with the cells therein.

27. The device of claim 21 wherein the tissue digestion chamber has an air burst filter near an air inlet of the tissue digestion chamber, the air burst filter

81 including a plurality of holes formed in a plate, wherein the air burst filter is configured to create bubbles from air passing through the plurality of holes of the air burst filter.

28. A method of isolating cells from biopsy tissue, the method comprising: inserting the biopsy tissue into a tissue digestion chamber; inserting one or more enzymes into the tissue digestion chamber; digesting the biopsy tissue to release the cells using the one or more enzymes; filtering a solution containing the cells through one or more sieves to separate tissue debris from the cells; passing the solution containing the cells through a waste filter membrane to retain the cells; and collecting the cells for delivery to a second device.

29. The method of claim 28 further comprising inserting a washing solution into the tissue digestion chamber to wash the biopsy tissue and the cells.

30. The method of claim 28 further comprising: passing the solution containing the cells into a cell concentration chamber; diluting the solution containing the cells within the cell concentration chamber; and passing the solution once diluted through the waste filter membrane to retain the cells within the cell concentration chamber but allow the solution to pass to or from the cell concentration chamber.

31. The method of claim 30 wherein diluting the solution containing the cells within the cell concentration chamber includes pumping a wash solution into a second chamber that fluidly communicates with the cell concentration chamber through the waste filter membrane.

82

32. The method of claim 31 wherein the pumping the wash solution includes pumping the wash solution into the cell concentration chamber.

33. The method of claim 32 wherein the solution without the cells diffuses across the waste filter membrane.

34. The method of claim 33 wherein the wash solution diffuses across the waste filter membrane and into the cell concentration chamber to form a suspension with the cells.

35. The method of claim 32 further comprising repeating pumping the wash solution into the second chamber that fluidly communicates with the cell concentration chamber through the waste filter membrane and further diffusing the wash solution across the waste filter membrane.

36. The method of claim 31 wherein at least a portion of the wash solution is from a cell sieve unit that performs the filtering the solution containing the cells through one or more sieves.

37. The method of claim 28 wherein inserting biopsy tissue into the tissue digestion chamber comprises: retaining the biopsy tissue in or on a biopsy holder; and inserting the biopsy holder and the biopsy tissue into the tissue digestion chamber.

38. The method of claim 28 wherein digesting the biopsy tissue includes injecting air into the tissue digestion chamber and producing air bubbles to agitate the biopsy tissue as it is being digested by the one or more enzymes.

39. The method of claim 38 wherein the air is injected into the tissue digestion chamber continuously at a flow rate between about 50 and about 300 ml/min.

83

40. The method of claim 38 wherein the air is injected via a pulse at a constant or variable airflow between about 50 and about 300 ml/min.

41. The method of claim 28 wherein digesting the biopsy tissue includes shaking the tissue digestion chamber to agitate the biopsy tissue as it is being digested by the one or more enzymes.

42. The method of claim 28 further comprising: controlling foaming in the tissue digestion chamber using one or more anti-foam agents.

43. The method of claim 42 further comprising siliconizing components including the tissue digestion chamber and the waste filter membrane with the one or more anti-foam agents.

44. The method of claim 28 wherein inserting one or more enzymes into the tissue digestion chamber includes inserting at least one of trypsin, dispase, collagenase and gentlyase.

45. The method of claim 44 wherein inserting one or more enzymes includes inserting two or more enzymes and each enzyme is inserted sequentially, and performing for each enzyme: passing one of the one or more the enzymes out of the tissue digestion chamber; and inserting a washing solution into the tissue digestion chamber to wash the biopsy tissue and released cells.

46. The method of claim 28 wherein the biopsy tissue is selected from skin, liver, umbilical cord, bone and other tissues and organs.

47. The method of claim 28 wherein the cells collected in the second device are in a single cell suspension and are used in in vivo, ex vivo, and in vitro applications.

84