WO2021003257A1

WO2021003257A1 - Methods and compositions for collecting and using placental tissue cells and placental blood cells

Info

Publication number: WO2021003257A1
Application number: PCT/US2020/040473
Authority: WO
Inventors: Rouzbeh R. Taghizadeh; Kyle Cetrulo
Original assignee: Auxocell Laboratories, Inc.
Priority date: 2019-07-01
Filing date: 2020-07-01
Publication date: 2021-01-07

Abstract

This disclosure provides a method of collecting placental tissue cells and placental blood cells and other components of the placenta using mechanical and/or enzymatic digestion of the placenta, wherein the method does not include a perfusion step. Compositions comprising the collected cells and/or other components of the placenta are provided. Methods of using placental tissue cells and placental blood cells (e.g., for stem cell transplantation) and methods of using other components of the placenta are also provided.

Description

METHODS AND COMPOSITIONS FOR COLLECTING AND USING PLACENTAL TISSUE CELLS AND PLACENTAL BLOOD CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional

Application No. 62/869,469, filed July 1, 2019, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND

[0002] The mammalian placenta is an organ of fetomaternal origin that plays an important role in fetal development, including enabling gas exchange, providing nutrients and removing waste products, and performing secretory and immunomodulatory functions. In addition, the placenta provides a heterogeneous population of cells useful in research and therapeutic applications, such as (1) placental tissue cells, including endothelial, epithelial, mesenchymal stem/stromal cells and (2) placental blood cells, including hematopoietic progenitor and hematopoietic stem cells.

[0003] Existing cell collection methods use perfusion to collect placental blood before collecting cells from placental tissue (see, e.g., U.S. Patent Nos. 8,329,468 and 7,045,148). However, inclusion of a perfusion step can be inconvenient and time consuming. Thus, there is a need in the art for improved methods for collecting placental tissue cells and placental blood cells from the placenta.

SUMMARY

[0004] The present disclosure relates to the discovery that both placental tissue cells and placental blood cells can be collected from the placenta without the need for a perfusion step. Placental tissue cells and blood cells can be collected in one process without the need to collect cells from the blood and the tissue separately.

[0005] Accordingly, the disclosure provides a method of collecting cells from placental tissue and placental blood without the need for a perfusion step. The method includes mechanically and/or enzymatically digesting the placental tissue and collecting the placental tissue cells and the placental blood cells into a container, e.g, a sterile container. [0006] In certain embodiments, the placental tissue is not enzymatically digested. In other embodiments, the tissue is enzymatically digested. In certain embodiments, the placental tissue is mechanically digested. In certain embodiments, the placental tissue is mechanically and enzymatically digested.

[0007] In certain embodiments, the method further includes a step of collecting extracellular matrix (ECM), ECM proteins and/or undigested tissue.

[0008] In certain embodiments, the placental tissue is mechanically digested using a tissue mincing tool. The tissue mincing tool includes a compartment for the placental tissue, a cutting surface at one end of the compartment, and a container ( e.g ., a sterile container). The cutting surface separates the compartment from the container such that the placental tissue that passes through the cutting surface is deposited within the container.

[0009] In certain embodiments, the method of collecting cells from a placenta containing the placental blood described herein further includes diluting the placental tissue after the mechanical and/or enzymatic digestion, for example, with a buffer. In certain embodiments, the method described herein further includes a filtering step, which removes undigested tissue from the digested placental tissue, and generates a filtrate that contains the placental tissue cells and placental blood cells.

[0010] Furthermore, in certain embodiments, the method comprises a sedimenting step.

In other embodiments, the method does not comprise a sedimenting step. In embodiments that include a sedimenting step, the sedimenting step can include sedimenting the filtrate formed by filtering the diluted digested placental tissue. In certain embodiments, the method includes re-suspending the sedimented cells and filtering the re-suspended cells.

[0011] In certain embodiments, the method includes isolating the placental tissue cells and/or the placental blood cells.

[0012] Compositions and cryopreserved compositions containing the collected cells, the ECM, ECM proteins and/or undigested tissue are also provided.

[0013] This disclosure also provides a method for performing a hematopoietic, mesenchymal stem and/or stromal cell transplant. The method includes transplanting cells collected by the methods described herein into a subject in need thereof, wherein the subject suffers from a disorder treatable by hematopoietic and/or mesenchymal stem/stromal cell transplant.

BRIEF DESCRIPTION OF THE FIGURES

[0014] Other features and advantages of the present invention, as well as the invention itself, can be more fully understood from the following description of the various

embodiments, when read together with the accompanying drawings, in which:

[0015] FIG. 1 provides an overview of exemplary procedural steps for collecting and isolating desired cells from the placenta, according to an embodiment of the invention.

[0016] FIG. 2 provides an overview of exemplary procedural steps for collecting and isolating desired cells from the placenta, according to an embodiment of the invention.

[0017] FIGS. 3A-3G depict procedures for collecting and isolating desired cells from the placenta.

[0018] FIGS. 4A-M provides fluorescent-activated cell sorting (FACS) results for placenta processed using the methods described herein. All histograms exhibit both isotype controls (left peak, blue) and the indicated marker expression (right peak, red or orange).

FIG. 4A shows a histogram for the full collection of cells isolated from human placenta.

FIG. 4B shows a histogram for cells stained with the viability dye 7-AAD (7- Aminoactinomycin D) to distinguish live and dead cells. The Y-axis is labeled“count” and the intervals across the Y-axis are labeled with 0, 500, 1.0K and 1.5K. The X-axis is labeled “Viability [7AAD] and the intervals across the X-axis are labeled with 10°, 10¹, 10², 10³, 10⁴, 10⁵ and 10⁶. FIG. 4C shows a histogram for cells stained with anti-CD29, anti-CD73 and anti-CD105 fluorescent antibodies. The Y-axis is labeled“Normalized to Mode” and the intervals across the Y-axis are labeled with“0, 20, 40, 60, 80 and 100.” The X-axis is labeled with“Isotype Control” and the markers used and the intervals across the X-axis are labeled with“10°, 10¹, 10², 10³, 10⁴, 10⁵ and 10⁶.” FIGS. 4D-G show histograms for cells stained with anti-CD90, anti-CD31, anti-CD146, and anti-CD326 fluorescent antibodies,

respectively. FIG. 4H shows a histogram for cells stained with anti-CD45, anti-CD34, anti- CD 1 IB, anti-CD 19, and anti -HLA- ABC fluorescent antibodies. FIGS. 4I-4M show histograms for cells stained with anti-CD45, anti-CD34, anti-HLA-ABC, anti-CDl lb, and anti-CD19 fluorescent antibodies, respectively. The Y-axis is labeled“Normalized to Mode” and the intervals across the X-axis are labeled with“0, 20, 40, 60, 80 and 100.” The X-axis is labeled with“Isotype Control” and the markers used and the intervals across the X-axis are labeled with“10°, 10¹, 10², 10³, 10⁴, 10⁵ and 10⁶.”

[0019] FIGS. 5A-M provide larger views of FIGS. 4A-M.

DETAILED DESCRIPTION

[0020] To provide an overall understanding of the invention, certain illustrative embodiments will now be described, including compositions and methods relating to collecting and using placental blood cells, placental tissue cells, and other placental components. However, it will be understood by one of ordinary skill in the art that the compositions and methods described herein may be adapted and modified as is appropriate for the application being addressed and that the methods described herein may be employed in other suitable applications. All such adaptations and modifications are to be considered within the scope of the invention. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and permutations.

[0021] The disclosure provides a method of collecting cells from a placenta, without prior removal of the placental blood, e.g ., by perfusion. The method includes mechanically and/or enzymatically digesting the placenta, and collecting the placental tissue cells and placental blood cells into a container, e.g. , a sterile container. The method further allows for the collection of extracellular matrix (ECM), ECM proteins, secretome components, and/or undigested placental tissue. The components collected from the placenta can be used for any suitable purpose, including in vitro and/or in vivo preclinical and clinical studies, cell transplantation (e.g, hematopoietic and/or mesenchymal stem/stromal cell transplantation), or administration of ECM, ECM proteins, and/or secretome components to a subject. The undigested placental tissue can be used as a seeding source for the further expansion of cells, e.g, mesenchymal stem cells (MSCs). The ECM, ECM proteins and/or secretome can be used, e.g, as a cell culture medium supplement.

[0022] The methods described herein allow for the simultaneous collection of placental tissue cells and placental blood cells in one procedure, without the need for a separate perfusion step to isolate placental blood cells. The discovery that both placental tissue cells and placental blood cells could be isolated in one procedure, without the need for a perfusion step, was surprising because prior to the instant discovery it was believed that, following the delivery and collection of a placenta, placental blood coagulated and a perfusion step was required for placental blood cells to be collected.

[0023] Further, collecting both placental tissue cells and placental blood cells together can be advantageous in certain applications. For example, it is believed that in a stem cell transplant, administration of hematopoietic stem cells (a type of placental blood cell) in combination with placental tissue cells ( e.g ., mesenchymal stem/ stromal cells and others) will increase hematopoietic stem cell engraftment and/or survival. Without wishing to be bound by the theory, it is believed that placental tissue cells therapeutically suppress the local immune environment during a stem cell transplant and/or increase the homing capacity (i.e., the ability to locate the appropriate target tissue) of the hematopoietic stem cells.

[0024] The terms“placenta” and“placental tissue” are used interchangeably herein. In certain embodiments, the term“placenta” or“placental tissue” includes the umbilical cord, amnion, and/or chorion. In certain embodiments, the term“placenta” or“placental tissue” does not include the umbilical cord.

[0025] The term“placental tissue cells” as used herein means any cell derived from placental tissue except for placental blood cells. For example, placental tissue cells can include endothelial cells, epithelial cells, mesenchymal stem/stromal cells (MSCs), trophoblastic stem cells, and pericytes. The term“placental blood cells” as used herein means any cell derived from placental blood, including for example, erythrocytes, hematopoietic stem cells and hematopoietic progenitor cells. Placental tissue can be obtained from any mammal having a placenta, including, for example, murines, simians, equines, bovines, porcines, canines, felines, and the like, and more preferably humans.

Mechanical and/or enzymatic digestion of placenta

[0026] Mechanical and/or enzymatic digestion of a placenta can be carried out using the following methods. In certain embodiments, the placenta is digested by a tissue mincing tool, which includes a compartment for the placental tissue, a cutting surface at one end of the compartment, and a sealed container. The cutting surface separates the compartment from the sealed container, such that the placenta passing through the cutting surface is deposited within the container. For example, the cutting surface may comprise a screen which acts as a cutting surface, and allows the digested placenta to pass through into the container. The cutting surface can be dimensioned to have an average cross-section of any size, e.g ., no greater than four square millimeters, no greater than one square millimeter, etc. The cutting surface can also include an automated cutting system. For example, the cutting surface may include semi-automatic scissors. The compartment for the tissue sample can incorporate one or more features to facilitate the application of a force to the tissue sample, impelling it toward the cutting surface (e.g., a screen) where it is cut and then passes through the cutting surface into the sealed container. For example, the tissue sample may be pressed toward the cutting surface, whereby a blade, or series of blades, cuts the tissue against the cutting surface. A portion of the compartment can be shaped to receive a solid member to press the tissue sample, or a shaft crank can be included for moving the cutting surface towards the tissue sample. Tissue mincing tools as described in U.S. Patent No. 8,893,995, incorporated by reference herein for all purposes, can be used in the methods described herein.

[0027] The sealed container may include at least one sealed access port permitting the sterile introduction of a fluid (e.g, a buffer, a cell culture medium, or water) into the container, and dilution of the mechanically digested placental tissue. An enzyme may also be injected into the sealed container, such that the enzyme enhances the digestion of the mechanically digested placental tissue. In certain embodiments, the enzyme does not come into contact with the mincer. The enzyme can be a protease, such as collagenase, hyaluronidase, trypsin, or dispase, separately or in combination. The tissue mincing tool can include a fluid conduit in communication with the sealed container, and a separator unit such as one or more filters (1000 microns to 1 micron) within the fluid conduit, such that the digested placental tissue is filtered and fragments larger than about 250 microns (e.g, fragments retained by a filter having a pore size of about 250 microns to 40 microns, referred to herein as“undigested tissue” or“undigested placental tissue”) can be retained in the sealed container. The tissue mincing tool can include a second sealed container for collecting the filtrate containing the placental tissue cells, placental blood cells, ECM, ECM proteins and secretome components. The tissue mincing tool can optionally include a fluid passageway in communication with the second sterile, sealed container, wherein the fluid passageway includes a cell capture zone. In some embodiments, the placental tissue cells and placental blood cells can be collected from the filtrate, e.g, by sedimentation, such that sedimented cells are concentrated in the cell capture zone. In some embodiments, the tissue mincing tool further includes a third sealed container in communication with the fluid passageway, wherein the sedimented cells can be resuspended with a buffer. Furthermore, the resuspended cells can be re-filtered, and re-sedimented.

[0028] FIG. 1 provides an overview of the procedural steps for an exemplary method of isolating and collecting cells. Generally, a placenta or portion thereof (“tissue”) can be initially minced using a tissue mincing tool by impelling the tissue sample toward the cutting surface of the tool, where the tissue sample is cut and then passes into a sterile, sealed container. The invention also provides optional methods of further digesting the tissue sample by exposing it to a chemical or an enzyme. For example, the mechanically digested placental tissue may be digested by injecting an enzyme into the container, such that the enzyme digests the mechanically digested placental tissue. The enzyme can be a protease, such as collagenase, hyaluronidase, trypsin, or dispase, separately or in combination. These steps are additionally or optionally incorporated into a method of separating mechanically and/or enzymatically digested placental tissue from any larger fragments (“undigested tissue,” as described above), for example, decanting, aspiration, sedimentation, or preferably, filtering. Optionally, the undigested placental tissue that is removed may be stored and/or used for other purposes such as a seeding source for the expansion of ex vivo cultured cells (e.g., stem cells). In some embodiments, the mechanically and/or enzymatically digested tissue, which can be viscous, is washed or diluted before a separating step. The separation of the placental tissue cells and/or placental blood cells from the mechanically and/or enzymatically digested tissue can be accomplished by sedimentation of the cells from a mixture containing the mechanically and/or enzymatically digested tissue. Although gravity sedimentation can be used, the sedimentation process can be accelerated by, for example, centrifugation. In certain embodiments, no separating step is performed and the digested tissue, cells, and liquid can be collected together. In certain embodiments, the placental tissue cells and/or placental blood cells are moved into a sterile container to be cryopreserved for later use.

[0029] In an embodiment, methods for separating mechanically and/or enzymatically digested tissue sample from undigested tissue may include two or more filtration steps as depicted in FIG. 2. For example, mechanically and/or enzymatically digested tissue sample may be subjected to multiple filtration steps using filters of varying sizes. In an embodiment, the mechanically and/or enzymatically digested tissue sample is initially subjected to a first filtration step using a large-pore filter of, e.g., about 1000 microns, 500 microns, about 250 microns, about 150 microns or about 100 microns, for removing coarse undigested tissue. Additionally, a second filtration step can be carried out to filter the eluate from the first filtration step using a small-pore filter of, e.g ., about 70 microns or about 40 microns, for removing additional contaminants such as collagen fibers. Filtering steps can be carried out using filter bags having an in-bag filter, as described herein.

[0030] The appropriate filter size (e.g., for an in-bag filter) can be selected according to the user’s objective, depending upon the size(s) of the material(s) to be collected. In certain embodiments, a filter size of about 1000 microns to about 1 micron is used. For example, in certain embodiments, a filter size of about 1000 microns to about 10 microns can be used to separate tissue from cells (generally about 7 microns to 9 microns). In certain embodiments, a filter size of about 1000 microns to about 500 microns, about 1000 microns to about 250 microns, about 1000 microns to about 100 microns, about 1000 microns to about 1 micron, about 500 microns to about 250 microns, about 500 microns to about 100 microns, about 500 microns to about 10 microns, about 500 microns to about 1 micron, about 250 microns to about 100 microns, about 250 microns to about 10 microns, about 250 microns to about 1 micron, about 100 microns to about 10 microns, or about 100 microns to about 1 micron is used. In certain embodiments, the filter size is about 10 microns, about 20 microns, about 30 microns or about 40 microns.

[0031] Because the mechanically and/or enzymatically digested tissue can be viscous, the tissue can be washed or diluted with an appropriate sterile solution (such as a buffered salt solution) at any stage in the process. For example, after the mechanically and/or

enzymatically digested tissue has been separated from the undigested tissue following the first filtration step, further washes can be performed to further cleanse the mechanically and/or enzymatically digested tissue before the second filtration step. Following multiple rounds of filtration, placental tissue cells and/or placental blood cells substantially free of placental tissue can be collected by sedimentation.

[0032] FIG. 3A depicts an exemplary procedure for collecting and isolating placental tissue cells and/or placental blood cells from a placenta. Prior to placing the placenta into a compartment, the placenta may be divided into smaller portions so that it will fit within the compartment. Depending upon the size of the placenta and of the compartment, the digestion and collection procedure may be performed repeatedly for each portion of the placental tissue so that the entire placenta can be digested. For example, a placenta may be about 400 ml to about 1000 ml, and may be divided up into 100 ml segments which can be added to the compartment one at a time until the entire placenta is processed, or a selected portion of the placenta is processed.

[0033] After being placed within a compartment, the placental tissue can be minced, parsed, or separated into even smaller portions. An advantage of mechanically digesting ( e.g ., mincing) the placenta before any enzymatic digestion is that the entire surface area of the tissue sample on which the enzyme can act is increased. The compartment may be fitted and attached to one port (e.g., an aperture) of a container (e.g, digestion bag) such that a placenta introduced into the compartment can directly pass through into the container. The

compartment may or may not be removably attached to the container. In other words, the compartment may be attached to the container, but in such a way that it can be removed from the container, or it may be permanently affixed to the container.

[0034] The container defines a sealed interior space that holds the mechanically digested placenta and fluids. The container may include sealed ports for introducing or dispensing materials and fluids into or from the container. For example, the container may include one or more injection ports for introducing fluids and one or more withdrawal ports for dispensing or suction fluids and materials from the container. Further, in an alternative embodiment, each of the injection ports and withdrawal port can be configured such that fluids and materials can only be moved in one direction to and from the container. Moreover, the ports can be disposed at an opposite end of the container from the compartment, though the ports can also be disposed along any portion of the perimeter of the container. In an embodiment, the ports are not removably secured to the container. Additionally or alternatively, syringes, air vents, capped air vents, or other devices that mate with a luer connection can be attached to the ports. All ports may be swabbable so that sterility is maintained.

[0035] In certain embodiments, placental tissue cells and/or placental blood cells are collected from the mechanically digested placenta without a perfusion step and without enzymatic digestion of the placenta. In other embodiments, the mechanically digested placenta is digested further by, for example, exposing it to a chemical or an enzyme. In an embodiment, the mechanically digested placenta may be digested further by an enzyme, for example, a protease, such as a collagenase, hyaluronidase, trypsin, or dispase, separately or in combination. The enzyme may be directly introduced into the container, such that the enzyme digests the mechanically digested placenta. For example, a syringe, or any other device that can house fluids, materials, or air, can be connected to the container (e.g, via a luer connection) and used to dispense, for example, a protease into the container to further digest the mechanically digested placenta. To enhance chemical digestion of the mechanically digested placenta, the container can be inverted to circulate the enzyme about the container. Depending on the rate of enzymatic breakdown of the mechanically digested placenta, the container can be placed at rest and the mechanically digested placenta can be incubated with the enzyme at 37 °C for a period of time, for example, for about one to three hours, though more or less time is contemplated, to digest the placenta. Additionally or alternatively, to assist in the incubation process, the container can optionally be periodically mixed with an orbital shaker or moved through a series of rollers or other compression-type device to assist in the breakdown of the placenta within the container. In an example in which the

mechanically digested placenta is about 100 mL, a user can inject about 10 mL of enzyme into the container, though more or less enzyme is contemplated. Once the mechanically digested placenta is also enzymatically digested, a digested placenta of about 110 mL results.

[0036] Following the mechanical and/or enzymatic digestion of the placenta, the digested placental tissue, which includes placental tissue cells, placental blood cells and ECM, ECM proteins and/or secretome components can be collected and used immediately or preserved, e.g ., cryopreserved. The collection of placental tissue cells and/or placental blood cells together with the ECM, ECM proteins, and/or secretome may advantageously provide support and nourishment for the cells. Without wishing to be bound by the theory, collection of all placental components together may act to maintain the natural environment of the cells, which may improve the condition of the cells, prevent deterioration, and provide for an improved population of cells having increased survival and/or engraftment upon transplant.

[0037] Alternatively, the components of the digested placental tissue can be separated. For example, placental tissue cells and/or placental blood cells can be separated from the ECM, ECM proteins, and/or secretome and from the undigested placental tissue. Each component may be collected separately and used immediately or preserved (e.g,

cryopreserved) for later use. In one embodiment, before the cells are separated from the mechanically and/or enzymatically digested placenta, any remaining fragments of undigested placental tissue are optionally removed to facilitate the subsequent purification of the cells. Depending on their size, undigested placental tissue can be removed by, for example, physical extraction, decanting, aspiration, sedimentation, or preferably, filtering. Optionally, the undigested placental tissue that is removed may be stored and/or used for other purposes such as a seeding source for the expansion of stem cells.

[0038] FIGS. 3A-3D illustrate various embodiments in which separation is achieved by filtration. Specifically, FIGS. 3A-3D depict a fluid passageway that connects the container holding the digested placenta to a filter unit which can be removably attached to the container. The filter unit may use a single filter, or a plurality of filters, optionally of decreasing size. Alternatively, a filter may be fitted and disposed in the container such that that the container is divided into two sub-spaces. The filter may be symmetrically or asymmetrically placed within the container. Additionally or alternatively, the filter may be fitted within a port, for example, a withdrawal port. The size of the filter can be about 1000 microns, about 500 microns, about 250 microns, about 150 microns, about 100 microns, about 70 microns, about 40 microns, about 10 microns, about 1 micron, or any range thereof, depending on the application. The digested placenta, which can be viscous, may be diluted prior to filtering so that the resultant diluted placenta can more easily move through the filter into downstream containers or components for further processing. Examples of diluting solutions include phosphate buffered saline (PBS), 5-20% human serum albumin, saline, plasmalyte, heta-starch, fresh plasma ( e.g ., autologous plasma), and allogeneic serum. In an embodiment, a syringe, or any other device that can house fluids, is used to dispense a diluting solution into the container via an injection port. In an example in which the digested placenta is about 400-1000 mL, a user can inject about 250 mL of a diluting solution into the container, though more or less solution is contemplated. As a result, the container holds about 650-1250 mL of a diluted, digested placenta. Following filtration, the eluate may be propelled, e.g., by vacuum, suction, or gravity, into a second sterile container (e.g, a wash/centrifugation bag) via a fluid passageway preferably regulated by line clamps (e.g, butterfly line clamps).

[0039] Isolating cells from diluted, mechanically and/or enzymatically digested placenta can be accomplished by various mechanisms. In certain embodiments, the placental tissue and/or placental blood cells are isolated from the diluted, mechanically and/or enzymatically digested placenta by filtration and/or by sedimentation. Although gravity sedimentation can be used, the sedimentation process can be accelerated by, for example, centrifugation. The present invention can include customized centrifuge buckets, inserts, and balance weights to ensure proper centrifuge of the system. In certain embodiments, sedimentation separates out all of the placental tissue and/or placental blood cells from the diluted, mechanically and/or enzymatically digested placenta. In certain embodiments, sedimentation separates out substantially all (i.e., more than 99.9%) of the placental tissue and/or placental blood cells from the diluted, mechanically and/or enzymatically digested placenta.

[0040] To facilitate cell collection, supernatants substantially or completely free of cells are optionally removed via an outlet port and a fluid passageway preferably regulated by line clamps. The supernatant may be removed by, for example, decanting or aspiration. In an example in which the second sterile container is a compressible bag, the supernatant may be decanted by physically pressing the bag. Alternatively, the supernatant may be removed, e.g ., by vacuum, suction, or gravity. Optionally, the supernatant can be removed into a waste container that is connected to the second sterile container through an outlet port and a fluid passageway regulated by line clamps. In an embodiment, the removed supernatant, which contains the ECM, ECM proteins and secretome may be stored and/or used for other purposes such as maintaining cells (in culture).

[0041] To collect placental tissue cells and/or placental blood cells, a small volume (e.g., 1-50 mL, for example, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL) of a diluting solution (e.g, phosphate buffered saline (PBS), 5-90% (e.g., 5-20%, 5-30%, 5-40%, etc.) human serum albumin, saline, plasmalyte, CryoStor® Base Medium (CSB), heta-starch, fresh plasma (e.g, autologous plasma), and allogeneic serum) can be added to resuspend the cell pellet which may be collected at the bottom of the second sterile container. As shown in the embodiment depicted in FIG. 3A, the second sterile container can have a bottom that is tapered to an angle sufficient to facilitate movement of the placental tissue cells and/or placental blood cells into a fluid passageway located at the bottom of the container, and optionally into a transfer container (e.g, transfer bag). In other embodiments (not shown), the second sterile container is a round-bottomed centrifuge bag. Alternatively, as depicted in FIG. 3B, placental tissue cells and/or placental blood cells may be moved into a fluid passageway located at the side of the container, and optionally into a transfer container. Movement of the cells out of the second sterile container and into the fluid passageway and optionally into a transfer container can be facilitated by gravity, vacuum or suction and may be regulated by line clamps.

[0042] If needed, the collected placental tissue cells and/or placental blood cells can be used immediately. Alternatively, the cells are cryopreserved for later use. To achieve long- term storage, cells can be transferred from the optional transfer container into a sealable, sterile container amenable to freezing ( e.g ., cryo-bag) or directly collected from the second sterile container into a freezable container for later use. Cryoprotectants are added to assist in storage and preservation of placental tissue cells and/or placental blood cells, and can include, for example, dimethyl sulfoxide (DMSO), albumin, and/or dextran, separately or in combination. Cryoprotectants may be added to the cells within the second sterile container following sedimentation. Alternatively, cryoprotectants may be added to and mixed with the cells within the optional transfer container or within the freezable container for long-term storage and later use.

[0043] In an embodiment, methods for separating mechanically and/or enzymatically digested placenta from undigested placental tissue may include two or more filtration steps as depicted in FIGS. 3C-3D. For example, mechanically and/or enzymatically digested placenta may be subjected to a first filtration step to remove coarse, undigested placental tissue.

Because the mechanically and/or enzymatically digested tissue can be viscous, the tissue can be washed or diluted with an appropriate sterile solution at any stage in the process. For example, after the mechanically and/or enzymatically digested tissue has been separated from the undigested tissue following the first filtration step, further washes can be performed to further cleanse the tissue prior to a second filtration step. In an embodiment, the second filtration step may utilize a smaller sized filter in order to remove contaminants such as collagen fibers from the mechanically and/or enzymatically digested tissue. Following the second filtration step, placental tissue cells and/or placental blood cells may be collected by sedimentation and moved into an optional transfer bag through a fluid passageway located either at the bottom of the container (FIG. 3C) or at the side of the container (FIG. 3D). Alternatively, the cells may be directly collected into a sterile cryo-bag for long-term storage and later use.

[0044] Additional exemplary processes for separating mechanically digested and/or enzymatically digested placenta are depicted in FIGS. 3E-3G. In each of these processes, the placenta is placed in a tissue mincer. In operation, the mincer forces the placenta through one or more cutting surfaces and deposits the finely minced tissue on the other side of the cutting surface(s). A saline bag is provided to permit the flushing of the placenta out of the mincer; typically, up to 500 mL of saline may be used for this purpose. When flushed from the mincer, the mechanically digested placental tissue may flow into an optional digestion bag (as shown), where the mechanically digested placental tissue may be enzymatically digested (as described with reference to FIGS. 3A-3D) prior to further processing. The optional digestion bag is in fluid communication with a dilution bag. Alternatively, if the mechanical digestion has obviated the need for any enzymatic digestion, the minced placental tissue can flow directly into a dilution bag. The mechanically digested placental tissue (whether or not subjected to enzymatic digestion) may be viscous. The dilution bag can be mechanically manipulated to encourage the mixing of the tissue and the saline. The dilution bag is also fitted with an optional injection port, permitting the injection of additional saline into the dilution bag as required.

[0045] The tissue suspension is then filtered, once the viscosity has been sufficiently reduced. As shown in FIGS. 3E-G, the suspension passes from the dilution bag into a filter bag having at least one in-bag filter. FIG. 3E depicts an embodiment with a single in-bag filter which retains particles larger than about 40-70 pm. FIG. 3F depicts an embodiment with a single in-bag filter which retains particles larger than about 150-250 pm. In FIG. 3F, the filtrate from the in-bag filter then passes through a second, in-line filter unit which retains particles larger than about 40-70 pm. FIG. 3G depicts an embodiment in which the filter bag contains two in-bag filters in succession, each in-bag filter having a surface area of at least 300 cm²; the first filter retains particles larger than about 500 pm and the second filter retains particles larger than about 100 pm. In each of FIGS. 3E-G, the filter bag includes a port permitting the removal of the retentate from the first filter. This retentate can optionally be used as a tissue explant for culturing cells.

[0046] The filtrates in FIGS. 3E-G pass into a centrifugation bag like those depicted in FIGS. 3A-D. Cells are separated from the suspension by sedimentation ( e.g ., by

centrifugation) and are concentrated in the bottom portion of the bag, or in a fluid

passageway connected to the bottom portion of the bag. The supernatant, which contains ECM, ECM proteins, and secretome, can be removed (e.g., by decanting, aspiration, vacuum, suction, or by compressing the bag) and collected for further use, e.g, for maintaining cells in culture.

[0047] To collect placental tissue cells and or placental blood cells, a small volume of a diluting solution (e.g, autologous plasma, DMEM, and/or phosphate buffered saline) can be added to resuspend sedimented cells. As shown in FIGS. 3E-G, the resuspended cells can pass from the centrifugation bag into a transfer bag, optionally after passing through a second filter bag, such as a second filter bag containing a filter having a surface area of at least 100 cm² and retaining particles greater than about 40 microns, as shown in FIG. 3G. The cells can be transferred to a cryobag and one or more cryoprotectants can be added, such as DMSO, albumin, and/or dextran, as described above for FIGS. 3A-D.

[0048] In addition to purified cells, the methods described herein also yield additional useful products. For example, when the cells are separated from the mechanically and/or enzymatically digested tissue, the remaining, cell-depleted tissue is a rich, sterile solution that can be used for maintaining cells ( e.g ., ex vivo , such as in culture). Further, any fragments of undigested tissue remaining after a digestion process can also be useful. For example, undigested placental tissue can be utilized as a seeding source for the expansion of mesenchymal stem cells.

[0049] In a specific embodiment of the methods described above, the placenta is mechanically and/or enzymatically digested using a closed system, e.g., the AC:Px System (AuxoCell Laboratories Inc., see, Taghizadeh et al. (2018) Cell Transplantation 27(1): 181- 193). The AC:Px System is a closed and single-use system to process solid tissue without the need for any enzymatic digestion. The AC:Px System consists of a closed mincer (i.e., semi- automated scissors) and a series of closed bags. The placenta can be processed by the AC:Px System to collect the placental tissue cells and placental blood cells, undigested placental tissue, and ECM, ECM proteins and/or secretome components.

Isolation of Placental Tissue Cells and Placental Blood Cells

[0050] After the collection (and optionally, purification) of the placental tissue cells and placental blood cells, various cell populations including trophoblasts, mesenchymal stem/stromal cells (MSCs), epithelial cells, myeloid cells, lymphoid cells, endothelial cells, pericytes, erythrocytes and hematopoietic stem/progenitor cells can be isolated separately or in combination.

[0051] For example, hematopoietic stem cells and various hematopoietic progenitor cells can be isolated by flow cytometry, which sorts cells based on their characteristic cell-surface protein expression, sizes and shapes. Table 1 below shows the cell-surface protein expression profile for a number of cells of interest. Table 1

[0052] Mesenchymal stem/stromal cells (MSCs) can be isolated using the methods known in the art, e.g ., according to the methods described in Pelekanos et al. (2016) J Vis EXP. (112): 54204; and Talwadekar et al. (2015) SCIENTIFIC REPORTS 5: 15784.

Trophoblastic, and more primitive stem cells from placenta can be isolated according Steigman et al. (2007) CuRR. PROTOC. STEM CELL BIOL. Chapter 1 : Unit 1E.2.

[0053] Collected cells can be tested for the presence or absence of markers of placental blood cells and placental tissue cells, which may include one or more of MSC markers (CD45, CD34, ELL A- ABC, HLA-DR, CDl lb, CD19, CD29, CD105, CD73, CD44, and CD90), hematopoietic/stem cell markers (CD34, CD133, CD45, CD33, CD38, and CD90), endothelial cell markers (CD31 and CD36), pericyte markers (CD146), and epithelial cell markers (CD326, CD46, CD 166, CD74), leukocytes (CD45, ELLA- ABC), macrophages (CD1 lb), and B-lymphocytes (CD 19), and any other markers of interest. Methods for Using Non-Digested Placental Components

[0054] Undigested tissue collected by the methods described herein can be used as a source to isolate various cell types, such as MSCs, using known methods in the art. For example, undigested placental tissue can be seeded into 100 mm culture dishes, which are coated with 1 mL of decellularized placental supernatant, along with 1 mL of RPMI-1640 medium (containing 20% MSC qualified FBS), 100 IU/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B, and 10 mg/mL ciprofloxacin, and placed in a 37 °C, 5% CO2 humidified incubator for 10 mins. In post-incubation, a 1 to 2 mL aliquot of the undigested tissue can be placed on the culture surface and spread by carefully swirling to disperse the tissue within the dish. The culture dishes can be placed in a 37 °C, 5% CO2 humidified incubator with the addition of 5 mL of medium dropwise (in order to prevent disruption of the seeded undigested placental tissue) to each dish every 4 days and incubated for a period of 10 to 14 days. After cells reached 70% to 90% confluency, the cells can be harvested, and tested for expression of cell surface markers CD29, CD45, CD105, CD73, CD90, and other markers of interest as described herein.

Methods for Isolating and Using ECM, ECM Proteins, and/or Secretome Components

[0055] The ECM, ECM proteins and/or secretome components collected by the methods described herein can include various growth factors ( e.g ., hepatocyte growth factor, neuron growth factor, endothelial growth factor, fibroblast growth factor, and insulin-like growth factor), proteins, lipids, nucleic acids, glycosaminoglycans, amino acids, vitamins, minerals and hormones (e.g., progesterone, and estrogen, human placental lactogen, and placental growth hormone). Each of the above components can be isolated separately or in

combinations from the collected ECM proteins/secretome and used immediately or preserved/cryopreserved for later use. For example, the ECM, ECM proteins and/or secretome components can be administered, e.g, via injection, to a subject.

Transplantation of Cells to a Subject

[0056] The placental tissue cells, the placental blood cells, any population of cells isolated from the placental tissue and placental blood cells (e.g, epithelial cells, endothelial cells, trophoblasts, MSCs, trophoblastic stem cells, pericytes, myeloid cells, lymphoid cells, erythrocytes and hematopoietic stem/progenitor cells), and/or any cells derived from the undigested placental tissue can be used for transplantation to a subject in need thereof, wherein the subject suffers from a disorder treatable by cell transplantation ( e.g ., stem and/or progenitor cell transplantation).

[0057] In certain embodiments, the subject suffers from leukemia, anemia, lymphoma, myeloma, an immune deficiency disorder, and/or a solid tumor, e.g., breast and ovarian cancer. In certain embodiments, the subject suffers from acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia,

amegakaryocytosis/congenital thrombocytopenia, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome, beta- thalassemia major, sickle cell disease, Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, familial erythrophagocytic lymphohistiocytosis, solid tumors, chronic granulomatous disease, mucopolysaccharidoses, or Diamond Blackfan anemia. In another embodiment, the subject is undergoing bone marrow ablative or non-myeolablative chemotherapy, or radiation therapy.

[0058] In some embodiments, the subject suffers from genetic diseases, e.g, B- thalassemia, sickle cell anemia, adenosine deaminase deficiency, recombinase deficiency, and recombinase regulatory gene deficiency. Genetic diseases associated with hematopoietic cells may be treated by transplantation of hematopoietic stem/progenitor cells to correct the genetic defect. Diseases other than those associated with hematopoietic cells may also be treated by the cell transplantation, wherein the disease is related to the lack of a particular secreted product such as a hormone, enzyme, interferon, factor, or the like. By employing an appropriate regulatory initiation region, inducible production of the deficient protein may be achieved, so that production of the protein will parallel natural production, even though production will be in a different cell type from the cell type that normally produces such protein. It is also possible to insert a ribozyme, antisense or other message to inhibit particular gene products or susceptibility to diseases, particularly hematolymphotropic diseases.

[0059] MSCs can be transplanted to a subject for treatment of connective tissue disorders, for example to promote the growth of connective tissues. The term connective tissue is used herein to include the tissues of the body that support the specialized elements, and includes bone, cartilage, ligament, tendon, stroma, muscle and adipose tissue. MSCs can be transplanted to a subject for regeneration of mesenchymal tissues which have been damaged through acute injury, abnormal genetic expression or acquired disease. In some embodiments, MSCs can be transplanted to a subject for treatment of autoimmune-, immune- or

inflammation-mediated diseases, such as multiple sclerosis, type 1 diabetes mellitus, graft versus host disease, osteoarthritis and inflammatory bowel disease, reviewed by Wang et al. (2016) Journal of Biomedical Science 23:76. MSCs can be transplanted to a subject for regeneration of cardiomyocytes in vivo (see, e.g., U.S. Patent No. 6,387,369). MSCs can also transplanted to a subject for treatment of pathologies of the central nervous system which are characterized by neuron loss, such as Parkinson's disease, Alzheimer's disease, stroke, and head trauma; or treatment of dysfunction in ganglioside storage or demyelinization, such as Tay-Sachs disease, G1 gangliosidosis, metachromatic leukodystrophy, and multiple sclerosis (see, e.g. , U.S. Patent No. 6,673,606). Furthermore, MSCs can be genetically engineered to express physiologically or pharmacologically active proteins for the transplantation. Genetic diseases or disorders which may be treated include, but are not limited to, cystic fibrosis, polycystic kidney disease, Wilson's disease, amyotrophic lateral sclerosis (or ALS or Lou Gehrig's Disease), Duchenne muscular dystrophy, Becker muscular dystrophy, Gaucher's disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, Charcot-Marie-Tooth syndrome, Zellweger syndrome, autoimmune polyglandular syndrome, Marfan's syndrome, Werner syndrome, adrenoleukodystrophy (or ALD), Menkes syndrome, malignant infantile osteopetrosis, spinocerebellar ataxia, spinal muscular atrophy (or SMA), and glucose galactose malabsorption.

[0060] Cells can be administered alone, or administered in the form of pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of cells, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, and combinations thereof.

[0061] The therapeutic compositions of the cells described herein are suitable for administration to a subject, including a human and a non-human animal. Such organisms preferably include, but are not limited to, mammals (e.g, murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0062] The therapeutically acceptable compositions suitable for administration to a patient may comprise one or more pharmaceutically acceptable carriers (additives) and/or diluents ( e.g ., pharmaceutically acceptable medium, for example, cell culture medium), or other pharmaceutically acceptable components. Pharmaceutically acceptable carriers and/or diluents are determined in part by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition. Accordingly, there is a wide variety of suitable formulations of therapeutic compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17^th ed. 1985).

[0063] The pharmaceutically acceptable carrier and/or diluent must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the therapeutic composition. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the therapeutic composition of the invention. For example, the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g, pregelatinized maize starch, polyvinylpyrrolidone and hydroxypropyl methylcellulose), a filler (e.g, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, and calcium hydrogen phosphate), a lubricant (e.g, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch,

polyethylene glycols, sodium benzoate, and sodium acetate), a disintegrant (e.g, starch, and sodium starch glycolate), or a wetting agent (e.g, sodium lauryl sulfate). Other suitable pharmaceutically acceptable carriers for the compositions of the present invention include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like. Such carrier solutions also can contain buffers, diluents and other suitable additives. The term "buffer" as used herein refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.

Examples of buffers envisioned by the invention include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water, and

normal/physiologic saline (0.9% NaCl).

[0064] In some embodiments, the cells are suspended in an appropriate diluent, at a concentration of from about 0.01 x 10⁶ to about 5x 10⁶ cells/ml for transplantation (e.g, about 0.01 x lO⁶ to about l x lO⁶ cells/ml, about 0.01 x lO⁶ to about 0.5x l0⁶ cells/ml, about 0.01 x lO⁶ to about 0.1 x lO⁶ cells/ml, about O.Ol x lO⁶ to about 0.05x 10⁶ cells/ml, about 0.05x l0⁶ to about 5x l0⁶ cells/ml, about 0.05 x lO⁶ to about l x lO⁶ cells/ml, about 0.05 x lO⁶ to about 0.5 x lO⁶ cells/ml, about 0.05 x lO⁶ to about O. l x lO⁶ cells/ml, about O. l x lO⁶ to about 5x l0⁶ cells/ml, about O.l x lO⁶ to about l x lO⁶ cells/ml, about O. l x lO⁶ to about 0.5x l0⁶ cells/ml, about 0.5x l0⁶ to about 5x l0⁶ cells/ml, about 0.5x l0⁶ to about l x lO⁶ cells/ml, about lxlO⁶ to about 5xl0⁶ cells/ml). Suitable excipients for such solutions are those that are biologically and physiologically compatible with the recipient, such as buffered saline solution. Other excipients include water, isotonic common salt solutions, alcohols, polyols, glycerine and vegetable oils. The composition for administration must be formulated, produced and stored according to standard methods complying with proper sterility and stability. The formulation should also suit the mode of administration.

[0065] Pharmaceutically acceptable carriers and/or diluents may be present in amounts sufficient to maintain a pH of the therapeutic composition containing the collected cells of between about 3 and about 10. In one aspect, the pH of the therapeutic composition is in the range from about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range from about 5 to about 9, from about 6 to about 9, or from about 6.5 to about 8. In some embodiments, the therapeutic composition comprises a buffer having a pH in one of said pH ranges. In some embodiments, the therapeutic composition has a pH of about 7.

Alternatively, the therapeutic composition has a pH in a range from about 6.8 to about 7.4. In still another embodiment, the therapeutic composition has a pH of about 7.4.

[0066] The compositions of the present invention ( e.g ., purified cells or ECM, ECM proteins, and/or secretome) can be administered parenterally, such as by systemic intravenous injection or injection directly to the intended site of activity. In some embodiments, the composition are administered via subcutaneous implantation. In some embodiments, the compositions are administered intramuscularly, intraarticularly, or intrathecally. In some embodiments, the cells are delivered to the site of desired treatment or therapy and can be targeted to a particular tissue or organ. In some embodiments, the compositions are administered together with other biologically active agents.

[0067] The dose of the cells to be administered can vary within wide limits and will need to be fitted to the individual requirements in each particular case. The number of cells used will depend on the weight and condition of the recipient and other variables known to those of skill in the art. The cells can be administered by a route which is suitable for the particular tissue or organ to be treated. For example, an effective amount of hematopoietic stem and/or progenitor cells and/or MSCs can be transplanted to engraftment of the cells in the recipient. In some embodiments, the amount is from about 0.01 ^c 10⁶ to about 5x 10⁶ cells/kg for transplantation ( e.g ., from about 0.01 ^c 10⁶ to about 1 ^c 10⁶ cells/kg, about 0.01 ^c 10⁶ to about 0.5x l0⁶ cells/kg, about O.Ol x lO⁶ to about O. l x lO⁶ cells/kg, about O.Ol x lO⁶ to about

0.05x l0⁶ cells/kg, about 0.05x l0⁶ to about 5x l0⁶ cells/kg, about 0.05x l0⁶ to about l x lO⁶ cells/kg, about 0.05x l0⁶ to about 0.5x l0⁶ cells/kg, about 0.05x l0⁶ to about O. l x lO⁶ cells/kg, about O. l x lO⁶ to about 5 ^c 10⁶ cells/kg, about O. l x lO⁶ to about 1 ^c 10⁶ cells/kg, about O. l x lO⁶ to about 0.5 x lO⁶ cells/kg, about 0.5 x lO⁶ to about 5x l0⁶ cells/kg, about 0.5x l0⁶ to about 1 x 10⁶ cells/kg, about lxlO⁶ to about 5xl0⁶ cells/kg). The MSCs may be administered concurrently with the hematopoietic stem and/or progenitor cells.

EXAMPLES

[0068] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention.

Collection of Placental Tissue Cells and Placental Blood Cells From Placenta

[0069] This Example describes the processing of human placenta using the mechanical AC:Px® System, which fractionates human tissue into its various components, including the native cells that reside within the tissue, as well as the extracellular matrix (ECM) proteins that provide support, growth factors, cytokines, various glycosaminoglycans (GAGs), and other signaling proteins to the native cells. The human placenta is a highly vascularized organ that contains native cells and ECM proteins from both the placental tissue and blood. It was discovered that using the AC:Px® System to mechanically breakdown human placenta (without the aid of traditional biochemicals) results in the isolation of both live tissue and blood cell types.

[0070] Placenta samples were collected upon delivery from normal full term pregnancies. All samples are obtained with written, informed consent in accordance with the ethical committee requirements. Once collected, the placenta was washed with antibiotic or antiseptic solution and placed in a sterile container and delivered to the laboratory for processing. The placenta was cut into 100 ml pieces using a sterile pair of scissors, and then placed in the AC:Px System (AuxoCell, Cambridge, MA, USA) for processing without prior perfusion of blood from the placenta. Briefly, placental tissue pieces were placed in the input chamber of the AC:Px Mincer with the output chamber filled with 0.9% sodium chloride saline. After subsequent mincing and washes with saline, the postminced placenta was transferred into the supplied series of AC:Px bag sets in order to filter and centrifuge the cells. Filtration took place in the AC:Px filter bag that filters using a 100 micron mesh, and subsequent centrifugation took place in the AC:Px centrifuge bag, clipped on a 97 mm blood bag centrifuge adaptor (Beckman Coulter) suspended, using the AC:Px centrifuge clip (AuxoCell). The cells were centrifuged for 10-20 mins at 500-750 g in an Allegra X15R (Beckman Coulter) benchtop centrifuge. After centrifugation, the supernatant containing ECM, ECM proteins, and/or secretome was decanted into the AC:Px filter bag, and the cell pellet which contained the placental tissue cells and placental blood cells altogether was collected. The cell pellet was resuspended in 50 ml decanted supernatant, and then filtered through the remainder of the AC:Px bag set that included a 40 micron filter bag to further remove the undigested tissue from the cell suspension. The supernatant and undigested placental tissue collected above were cryopreserved for later use.

[0071] The resuspended cell pellet, containing isolated native cells from a human placenta, was analyzed using fluorescent-activated cell sorting (FACS), with the full collection of placental cells shown in FIGS. 4A and 5A. Cells were stained with the viability dye 7-AAD (7-Aminoactinmycin D) to distinguish live and dead cells (FIG. 4B, FIG. 5B). Cells were also stained with various fluorescent-tagged antibodies to exhibit the presence of both live tissue FIG. 4C-G; FIG. 5C-G) and blood (FIG. 4H and 5H) cell types. Tissue cell types isolated from human placenta include: mesenchymal stem/stromal cells (CD29, CD73, CD105, CD90) (FIG. 4C and 4D and FIG. 5C and 5D), endothelial cells (CD31) (FIG. 4E and 5E), perivascular/pericyte cells (CD146) (FIG. 4F and 5F), epithelial cells (CD326) (FIG. 4G and 5G). In addition, live native blood cells (FIG. 4H and 5H) were isolated from human placenta, including human leukocytes (CD45, HLA-ABC) (FIG. 41 and 4K; FIG. 51 and 5K), hematopoietic progenitor cells (CD34) (FIG. 4J and 5J), macrophages (CD1 lb) (FIG. 4L and 5L), and B-lymphocytes (CD 19) (FIG. 4M and 5M). All histograms exhibit both isotype controls (left peak, blue) and the indicated marker expression (right peak, red or orange). [0072] This example shows that mechanical digestion of human placenta using the AC:Px® System allows for the collection of both native placental tissue cells and placental blood cells.

INCORPORATION BY REFERENCE

[0073] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0074] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS:

1. A method of collecting cells from placental tissue and placental blood, the method comprising:

(a) mechanically and/or enzymatically digesting the placental tissue and

(b) collecting the placental tissue cells and the placental blood cells from the placental tissue into a container;

wherein the method does not comprise a perfusion step.

2. The method of claim 1, wherein the placental tissue is not enzymatically digested.

3. The method of claim 1, wherein the placental tissue is enzymatically digested.

4. The method of any preceding claim, wherein the placental tissue is mechanically digested.

5. The method of any one of claims 1, 3, and 4, wherein the placental tissue is mechanically and enzymatically digested.

6. The method of any preceding claim, the method further comprising collecting

extracellular matrix (ECM) and ECM proteins.

7. The method of any preceding claim, the method further comprising collecting undigested tissue.

8. The method of any preceding claim, wherein the placental tissue is mechanically digested using a tissue mincing tool comprising: a compartment for the placental tissue;

a cutting surface at one end of the compartment; and

the sterile container,

wherein the cutting surface separates the compartment from the sterile container such that the placental tissue that passes through the cutting surface is deposited within the sterile container.

9. The method of any preceding claim, the method further comprising diluting the digested placental tissue.

10. The method of any preceding claim, the method further comprising a filtering step.

11. The method of claim 10, further comprising diluting the digested placental tissue, wherein the filtering step comprises removing undigested tissue by filtering the diluted digested placental tissue.

12. The method of any preceding claim, the method further comprising a sedimenting step.

13. The method of claim 12, wherein the sedimenting step comprises sedimenting the filtrate formed by filtering the diluted digested placental tissue of claim 10.

14. The method of claim 13, further comprising re-suspending sedimented cells and filtering the re-suspended sedimented cells.

15. The method of any one of claims 1-11, wherein the method does not comprise a sedimenting step.

16. The method of any preceding claim, the method further comprising isolating the placental tissue cells and/or the placental blood cells.

17. A composition comprising the cells, ECM and ECM proteins, and/or undigested tissue collected according to the method of any preceding claim.

18. A method of performing a hematopoietic and/or mesenchymal stem cell transplant, the method comprising transplanting the composition of claim 17 into a subject in need thereof, wherein the subject has a disorder treatable by hematopoietic and/or mesenchymal stem/stromal cell transplantation.

19. A cryopreserved composition comprising the composition of claim 17, wherein the composition has been cryopreserved.