WO2017132358A1 - Methods for isolation and expansion of umbilical cord mesenchymal stem cells - Google Patents

Abstract

Methods directed to the isolation and expansion of umbilical cord mesenchymal stem cells from umbilical cord tissue are disclosed. The methods are particularly suited for processing whole tissues or whole tissue sections, without dissection or extraction of blood vessels from the umbilical cord tissue. The methods comprise a closed processing system for disruption wherein digestion and dissociation to isolate the umbilical cord mesenchymal stem cells occurs in the same container, so that opportunities for contamination are reduced.

Description

METHODS FOR ISOLATION AND EXPANSION OF UMBILICAL CORD

MESENCHYMAL STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of U.S. Provisional Patent Application

Serial No. 62/287,324, filed January 26, 2016, entitled CELLS AND BIOLOGICS FOR REGENERATIVE MEDICINE AND TISSUE ENGINEERING, incorporated by reference in its entirety herein. BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is generally directed to methods of isolating and expanding umbilical cord mesenchymal stem cells (UC-MSCs), particularly for use in clinical trials. Description of the Prior Art

Mesenchymal stromal cells (MSCs) play an important role in regenerative medicine, for cell therapy or tissue engineering. This importance is based on properties of these cells. MSCs have the capacity to differentiate to osteoblasts, adipocytes and chondrocytes, which classifies MSCs as multipotent stromal cells. MSCs may modulate the immune system, and enable tissue repair by secretion of growth factors, cytokines, and other signaling molecules into the medium. The immune properties of MSCs give these cells an important role to treat immunological disorders, such as graft-versus-host-disease.

MSCs are found in other tissues beside the marrow cavity, for example, they can be found in blood or adipose tissue, dermis, muscle, dental pulp, umbilical cord blood, placenta, perivascular areas, amniotic fluid, and tissues surrounding the umbilical cord vessels, called the Wharton's jelly. The advantage of isolation of MSCs from the umbilical cord is that collection is safe and painless to mother and child, in contrast to the invasive and painful extraction of MSCs from the bone marrow. While there are advantages to the choice of human umbilical cord mesenchymal stromal cells (hUC MSCs) as a MSC source, there are distinct challenges to using this source, which include the lack of standardized method for isolating, expanding and validating hUC MSCs.

The International Society for Cellular Therapy (ISCT) provides three minimal criteria to identify MSCs. First, MSCs must be tissue culture plastic-adherent when maintained in standard culture conditions. Second, they express specific surface antigens CD 105, CD73 and CD90 and they do not highly express markers of the hematopoietic lineage such as CD45, CD34, CD14, CDl lb, CD79a, CD19 or HLA class II. The third criterion is that MSCs must be capable to differentiate to osteoblasts, adipocytes and chondrocytes in vitro.

The acceleration of stem cell and regenerative medicine clinical trials, and MSC trials in particular, has produced a renewed effort to standardize production and characterization of MSCs in GMP compliant SOPs. Umbilical cord MSCs have a number of advantages, which suggest that they might be an important source for allogeneic MSCs for cellular therapy, and, as indicated by trends in MSC clinical trials worldwide, this MSC source is a needed one.

Several protocols for isolation of MSCs from different parts of the umbilical cord have been published. However, these prior art protocols require dissection of different portions of the umbilical cord and a variety of methods to enrich MSCs from the primary isolation population. This contributes to variation in the number of cells in the primary isolation, and their ability to undergo expansion in culture.

SUMMARY OF THE INVENTION

Methods of producing isolated umbilical cord mesenchymal stem cells from umbilical cord tissue are described herein. The methods generally comprise incubating umbilical cord tissue with a digestive enzyme solution to yield digested umbilical cord tissue. The digested umbilical cord tissue is then dissociated to yield a dissociated umbilical cord tissue solution comprising umbilical cord mesenchymal stem cells dispersed in the remainder of the umbilical cord tissue (e.g., tissue debris, etc.). The umbilical cord mesenchymal stem cells are separated from the dissociated umbilical cord tissue solution to yield separated umbilical cord mesenchymal stem cells, which are then suspended in a suspension comprising a quantity of xenogen-free culture medium enriched with human platelet lysate and red blood cell lysing solution to remove red blood cell contaminants. The umbilical cord mesenchymal stem cells are then isolated from the culture medium and red blood cell lysing solution, to yield isolated umbilical cord mesenchymal stem cells.

A xenogen-free culture medium is also described herein. The culture medium generally comprises glucose, glutamine, human platelet lysate, and heparin. BRIEF DESCRIPTION OF THE DRAWINGS

Figure (Fig.) 1 is a schematic of an isolation method in accordance with the present invention;

Figs. 2A-2C are graphs showing the effect of various experimental variables on UC-MSCs isolation;

Figs. 3A-3F are graphs showing the effect of human platelet lysate concentration on cell expansion;

Figs. 4A-4D are a photographs showing differentiation and colony forming unit fibroblast results for the validation of UC-MSCs and Fig. 4F is a graph showing CFU-F efficiency per HPL enrichment;

Figs. 5A-5E are graphs showing histograms of flow cytometry, wherein solid fill overlay represents the test sample, diagonal line filled overlay represents the isotype control;

Fig. 6 is a graph showing colony forming efficiency;

Fig. 7 is a graph showing growth kinetic of hUC MSCs from two different donors (female: black circles; male: white squares) in static 12-well-culture plate in 1 mL DMEM LG medium with seeding density of 8000 cells cm^"2;

Figs. 8A-8B are graphs showing the metabolite profile of hUC MSCs from two different donors (female: squares, male: circles) in static 12-well-culture plate in 1 mL DMEM LG medium with seeding density of 8000 cells- cm^"2. A: grey full - glucose, white - lactate, B: black - glutamine, grey empty - ammonium, dotted line: glutamine thermal decay;

Fig. 9 is a graph showing occupancy and confluency of five different microcarriers after 72 h culture time in ultra-low attachment 6-well plates with 0.0375 g of microcarriers in 1 mL DMEM LG medium at a seeding density of 8000 cells- cm^"2;

Fig. 10 is a pair of photographs showing SybrGreen stained hUC MSCs on HCl-treated Glass-coated microcarriers (left) and Plastic Plus microcarriers (right) after 3 days of culture in 1 mL DMEM medium in an ultra-low attachment well plate at a seeding density of 8000 cells- cm^"2 (scale bar= 1000 μπι);

Fig. 11 is a graph showing growth kinetic of hUC MSCs on Plastic Plus microcarriers in spinner flasks in 100 mL DMEM LG medium with seeding density of 8000 cells-cm^"2 (female: white squares; male: black circles);

Figs. 12A-12B are graphs showing metabolite profile of hUC MSCs from a male donor in a dynamic spinner culture with 25 g-L^'1, 100 mL DMEM LG medium and a seeding density of 8000 cells- cm^"2. A: grey full - glucose, white empty - lactate, B: black full - glutamine, grey empty - ammonium;

Fig. 13 is a photograph showing SybrGreen stained hUC MSCs on Plastic Plus microcarriers after 9 days of culture in 200 mL DMEM medium in a spinner culture at a seeding density of 8000 cells cm^"2;

Fig. 14 is a series of photographs showing differentiated hUC MSCs; left: adipocytes stained with Oil Red after 21 days; middle: osteocytes stained with Alizarin Red; right: chondrocytes stained with Safranin after 22 days;

Figs. 15A-15E are graphs showing histograms of flow cytometry analysis;

Fig. 16 is a graph showing the results of CFU-F analysis; and

Fig. 17 is an image showing the karyotype analysis of hUC MSCs at passage 5.

DETAILED DESCRIPTION

Methods in accordance with embodiments of the present invention are generally directed to the isolation and expansion of UC-MSCs from umbilical cord tissue. The umbilical cord tissue is obtained from umbilical cords collected from neonates after vaginal delivery or Caesarian- section births, and from either male or female donors. However, in preferred embodiments, the umbilical cords are collected from vaginal births. The methods described herein are suitable for use with human tissues, as well as non-human mammals, such as pigs, horses, dogs, cats, rats, mice, rabbits, monkeys, apes, and the like.

Generally, umbilical cords can be stored, for example in a sterile saline solution after collection. Preferably, the umbilical cord is processed according to methods of the invention within at least 5 days, more preferably within at least 4 days from collection. In other embodiments, however, umbilical cords or umbilical cord tissues that have been preserved (e.g., cryopreserved) can be used a greater time period after collection. The collected umbilical cords may be prepared by rinsing any surface blood with buffered saline and/or antibiotic-antimycotic solution, and then treating with an antiseptic solution, such as a povidine-iodine solution. The cord may then be cut into smaller sections, for example about 1 cm in length, about 2 cm in length, or about 3cm in length, for easier handling and processing. The cord or cords section(s) can then be rinsed until no further surface blood or blood clots are visible. The umbilical cord sections may be further reduced, for example by cutting or mincing into smaller pieces, depending on the size of the equipment being used. However, the present invention advantageously avoids the contamination risks associated with prior art methods, which require the removal of umbilical cord blood vessels prior to further processing. Therefore, in certain embodiments of the present invention, the blood vessels are not removed from the umbilical cord tissue prior to subsequent processing the umbilical cord tissue to isolate the UC-MSCs. That is, methods of the invention are particularly suited for processing whole tissues or whole tissue sections, without dissection or extraction of blood vessels from the umbilical cord tissue. Thus, "whole" tissues, according to the invention, denotes the fact that the umbilical cord interstitial and connective tissue remains intact along with its blood vessels characteristic of in vivo tissue, and is not decellularized, or otherwise disaggregated prior to being processed, with the understanding that the term "whole" is not intended to imply that a full and intact umbilical cord is necessarily processed. Indeed, the invention particularly contemplates processing smaller segments of umbilical cord in the method.

The umbilical cord tissue is then contacted with a digestive solution for disaggregation of the tissue to break down the intercellular network and connective tissue in the cord. In certain preferred embodiments, a digestive enzyme solution is used. A variety of commercially-available digestive enzyme solutions may be used. Exemplary digestive enzyme solutions include collagenase, hyaluronidase, DNase, elastase, papain, protease type XIV, and trypsin. In one or more embodiments, the digestive enzyme solution is a trypsin-based enzyme solution, a collagenase-based enzyme solution, a hyaluronidase-based enzyme solution, or a mixture thereof. In certain embodiments, a single digestive enzyme solution is used. In other embodiments, the umbilical cord tissue is sequentially contacted with two or more digestive enzyme solutions. For a collagenase-based enzyme solution, a concentration of from about 100 U/mL to about 1,000 U/mL, preferably about 250 U/mL to about 750 U/mL, and more preferably from about 500 to about 600 U/mL is used. For a hyaluronidase-based enzyme solution, a concentration of from about 0.01 mg/mL to about 10 mg/mL, preferably about 0.1 mg/mL to about 5 mg/mL, and more preferably from about 0.5 to about 2 mg/mL is used. For trypsin-based enzyme solutions (including trypsin- EDTA solutions), a concentration of from about 0.01% to about 1.0%, and more preferably from about 0.025%) to about 0.5% is used. In certain other embodiments, a chemical digestive solution may be used.

The tissue may be contacted with the digestive solution in a variety of containers to yield digested umbilical cord tissue. Preferably, the tissue is contacted with the digestive solution in a container suitable for subsequent incubation. More preferably, the container is also suitable for subsequent mechanical dissociation. That is, an additional advantage of the invention is a closed processing system for disruption wherein digestion and dissociation to isolate the UC-MSCs occurs in the same container, so that opportunities for contamination are reduced.

The umbilical cord tissue and digestive enzyme solution are incubated for a time period of less than about 10 hours, more preferably less than about 5 hours, even more preferably from about 2 to about 5 hours, and most preferably from about 3 to about 4 hours. While longer processing may increase yield, viability tends to decrease over time, as does attachment and expansion efficiency of cells, which would reduce the overall yield of useful cells. It will be appreciated that the incubation time will depend upon the concentration of the digestive solution, as well as the temperature. The incubation is carried out at a temperature from about 30°C to about 45°C, more preferably from about 35°C to about 40°C, and most preferably about 37°C.

After incubation, the digested umbilical cord tissue is mechanically dissociated to physically break up the interstitial and connective tissue, blood vessels, and the like, and dislodge individual components of the tissue to yield a dissociated umbilical cord tissue solution comprising the UC-MSCs. The mechanical dissociation may be performed by hand, but may also be carried out, for example, using a commercially-available tissue dissociation device, such as MACS® by Miltenyl Biotec.

The UC-MSCs are then separated from the dissociated umbilical cord tissue solution. Various separation or isolation techniques can be used, including centrifugation, density gradient centrifugation, as well as filtering or sieving, to separate the UC-MSCs from remaining tissue debris and extracellular components, etc. The cells are then pelleted via centrifugation, and then resuspended in suspension medium comprising a quantity of xenogen-free culture medium and red blood cell lysing solution to remove red blood cell contaminants. As used herein, "xenogen-free" refers to a culture medium having less than 1% by weight of substances originating outside of the umbilical cord donor species' (e.g., human) body. For example, fetal bovine serum is an example of a xenogen that is preferably excluded from the culture medium.

The culture medium is preferably a simplified medium formulation, comprising glucose and glutamine, for example Dulbecco's Modified Eagle Medium (DMEM), which generally comprises glucose (low or high) and glutamine (e.g., Gibco™ GlutaMAX™), as well as human platelet lysate (e.g., pooled human platelet lysate), and heparin. In certain preferred embodiments, the medium further comprises an antibiotic-antimycotic component, which reduces contamination risk. The presence of human platelet lysate provides improved cell growth and expansion over prior art mediums. Thus, in certain embodiments, the culture medium comprises from about 2% to about 30% by volume, preferably from about 5% to about 15% by volume, and most preferably about 10%) by volume of human platelet lysate. As indicated above, the medium will generally comprise some quantity of glucose and glutamine, although in certain embodiments no glucose and/or no glutamine is present in the medium. When present, the glutamine is generally present at a concentration of 0 mM to about 6 mM. The heparin is generally present in the culture medium at a level of 0 U to about 4 U. The culture medium used in embodiments of the present invention has a number of advantages over prior art UC-MSC culture mediums. For example, prior art mediums have generally comprised 10 or more components, which caused them to be expensive and impractical for Good Manufacturing Practice (GMP) manufacturing. The culture mediums used in certain preferred embodiments of the present invention have fewer components, which reduces cost and regulator overhead for GMP. Thus, in certain embodiments, the culture medium consists essentially of glucose, glutamine, human platelet lysate, and heparin. The red blood cell lysing solution removes any remaining red blood cell contamination from the UC-MSCs.

The UC-MSCs are then isolated from the culture medium and red blood cell lysing solution. Prior to this separation, a buffered saline solution may be added to the suspension and mixed. The UC-MSCs may be separated, for example, by filtering and/or centrifugation. In certain embodiments, the resulting isolated UC-MSCs can be pelleted and resuspended in a solution comprising a (second) quantity of the xenogen-free culture medium comprising human platelet lysate described above, for example for further testing or processing. Advantageously, the isolation methods described herein are capable of isolating at least about 10 times the amount of UC-MSCs over prior art isolation methods (see Fig. 2A, described below). For example, the methods described herein are capable of isolating at least about 100,000, preferably at least about 200,000, live cells per cm of umbilical cord tissue. Similarly, the methods described herein are capable of isolating at least about 100,000, preferably at least about 200,000, live cells per gram of umbilical cord tissue.

In order to provide increased availability of UC-MSCs for clinical trials, in certain embodiments the present methods further comprise expanding the isolated cells. In certain such embodiments, the separated UC-MSCs are added to a tissue culture plate (i.e., plated) or seeded on a microcarrier (i.e., seeded on microcarrier beads) comprising a quantity of xenogen-free culture medium comprising human platelet lysate. Microcarriers are small beads with a density slightly higher than medium so they are easily suspended in a moving fluid column. The sphere- shaped beads provide advantageous surface area to volume, and thus they can be an efficient means to increase surface area for attached cells to group upon. The size, density, and surface properties of the bead (e.g., charge, roughness, porosity, ligands, or coatings) are important physical properties which influence cell attachment and the ability to remain suspended in a fluid column by agitation. When a tissue culture plate is used, the cells can be plated using known techniques at a level of from about 5,000 to about 20,000 live cells per cm² and preferably from about 10,000 to about 15,000 live cells per cm² on the plate. When a microcarrier is used, the cells can be seeded using known techniques at a density of about 1,000 cells cm^"2 to about 20,000 cells-cm^"2, preferably about 5,000 cells cm^"2 to about 10,000 cells cm^"2, and more preferably about 8,000 cells cm^"2. In certain preferred embodiments, the microcarrier comprises a modified polystyrene or cross-linked polystyrene surface. In certain such embodiments, the surface may further comprise a cationic charge, or be coated with high silica glass or recombinant RGD-containing protein. In certain embodiments, cross-linked polystyrene microcarriers with cationic surface charge are particularly preferred.

After plating or seeding, the UC-MSCs are incubated, preferably at a temperature of from about 30°C to about 45°C, more preferably from about 35°C to about 40°C, and most preferably about 37°C. In certain embodiments, the cells are seeded on a microcarrier in a dynamic system, such as a spinner flask or bioreactor. In such embodiments, a stirrer or other means of agitation may be used to maintain the microcarrier beads in suspension and encourage cell growth. The cells are generally allowed to incubate until the culture achieves at least about 70% confluency (preferably from about 80% to about 90% confluency) and are then transferred to a second tissue culture plate or microcarrier comprising the culture medium described herein. This incubating- transfer procedure may be performed for multiple passages until the desired cell yield is achieved. This method is capable of achieving significantly higher cell viability and overall yield over prior art methods. Moreover, it will be understood that a combination of tissue culture plates and microcarriers may be used in the methods described herein (i.e., cells in a plate may be transferred to a microcarrier, and cells on a microcarrier may be transferred to a plate).

Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.

As used herein, the phrase "and/or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

EXAMPLES

The following examples set forth experiments for isolating and expanding UC-MSCs. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.

EXAMPLE I

This example is directed to an isolation method that decreases contamination risk and isolation time and increases the yield of MSCs. The method herein also describes a simplified medium that provides for robust expansion of MSCs, is xenogen-free, and is suitable for clinical manufacturing.

Materials and Methods

Umbilical Cords. Discarded, anonymous human tissue with all identifying linkages broken were used (IRB #5189). Tissue processing was performed inside a biological safety cabinet (BSC) in a BSL2 laboratory using universal precautions per Occupational Safety and Health Administration (OHSA) recommended blood borne pathogens containment described in 29 CFR. 1910.1030.

24 umbilical cords (11 females and 13 males) were used; umbilical cords from vaginal births or Caesarian-section births were used. The umbilical cords were stored in sterile tissue sample containers in saline solution at 4°C until use. Here, isolation procedures were performed within 4 days after birth. To randomize the treatment effects, no prescreening was performed and cord samples (biological replicates) were randomly assigned to each experimental variable.

Isolation strategy. The isolation method performed herein was aimed at decreasing contamination risk, increasing yield, and improving GMP compatibility. For each umbilical cord (the biological unit), eight randomly selected 1 cm length samples were used to test the effect on the experimental variables identified. Two to four variables were evaluated per cord using technical duplicates, and the results were averaged for each experimental variable per biological unit for comparisons. First, mechanical disruption of the tissue was tested using a Miltenyi GentleMACS Dissociator (#130-093-235) using preprogrammed settings A, B, C, D, and E, (which corresponds to weakest to strongest dissociation). Next, tissue dissociation conducted before or after enzymatic digestion was tested. Then, the effect of mincing the tissue samples was compared to tissue dissociation using the GentleMACS Dissociator. Next, the effect of filtering using ΙΟΟμπι cell strainers (FisherBrand # 22-363-549) and 60μπι SteriFlip tubes (Millipore # SCNY00060) was tested. Lastly, the concentration of enzyme was varied to determine the effect on yield. The technical duplicates or triplicates were averaged for each variable per cord sample. Each procedural variable was evaluated using at least three different cord replicates. Decision making strategy was designed-based using process yield (more live cells) or increasing process efficiency (reducing number of processing steps, reducing time, or reducing contamination risk).

Final isolation method. A schematic of the method is shown in Fig. 1. Umbilical cords were rinsed to remove surface blood using 37°C DPBS which had 1% Antibiotic-Antimycotic (Dulbecco's Phosphate Buffered Saline, Life Technologies #14190-250; Antibiotic- Antimycotic, Life Technologies #15240-062). The cords were then treated with 0.5% Betadine (Dynarex, Providium Iodine Solution, #1416) in DPBS for 5 minutes at room temperature. Inside the biological safety cabinet (BSC), the cord was cut into 1 cm lengths, and rinsed repeatedly with 3 volumes DPBS until no further surface blood could be seen. Each 1 cm length of tissue was cut into four equal size pieces and placed into a Miltenyi Biotech Dissociator C-Tube (Miltenyi #130- 096-334). The tissue weight was calculated by subtracting the tare weight of the C-tube and 9 mL of enzyme solution was added. The C-tubes were placed into a Miltenyi Dissociator, processed using program C and incubated for 3-3.5 hours at 37°C with constant 12 rpm rotation. Following the 3-3.5 hour incubation, the tissues were dissociated using program B and filtered through 60 μηι SteriFlip filter (Millipore #SCNY00060) to remove tissue debris. The cells were pelleted by centrifugation at 200x g for 5 minutes at room temperature, and the supernatant was discarded. The cells were suspended in 0.5 mL of growth media, and 0.5 mL RBC lysing solution (Sigma' s RBC lysis solution, #R7757-100ML) was added to remove red blood cell contamination. The cells were mixed gently for one minute followed by addition of 8 mL of DPBS. Cells were centrifuged at 200x g for 5 minutes at room temperature, and the supernatant was discarded. The cells were suspended in lmL of media and the number of live cells was determined using a Nexcelom Auto2000 cellometer (Immune cells program, low RBC) following ViaStain AOPI (acridine orange and propidium iodide) viability staining (Nexcelom cat #CS2-D106-5ML). Cells were plated at 10,000-15,000 live cells per cm² on tissue culture treated plastic (CytoOne 6 well plates, #CC7682-7506).

MSC Expansion. Previous work by this lab using a MSC expansion medium was the standard used for comparison. Since prior mediums contain more than 10 components, one goal of this experiment was to reduce the number of medium components while maintaining the MSC attachment at isolation/ startup, and maintaining MSC expansion, CFU-F efficiency, tri-lineage differentiation potential, MSC surface marker expression, and cellular morphology similar to or better than the prior art. Low glucose Dulbecco's Modified Eagle's Medium (DMEM Life Technologies cat#14190) supplemented with 1% Glutamax (Life Technologies cat #35050), 1% Antibiotic- Antimycotic, and with either 2, 5 or 10% by volume with pooled human platelet lysate (HPL, HPL pooled from more than 25 outdated platelet donors) and 4 Units/mL heparin was tested. The cells were plated at 10,000-15,000 cells per cm² in CytoOne flat bottom tissue culture treated 6 well plates and expanded for 5 passages. Cells were incubated and grown as a monolayer at 37°C, 5% CO2 and 90% humidity (Nuaire AutoFlow 4950 or Heracell 150i). Once the cells reached approximately 80-90% confluence they were lifted and plated in fresh medium. To lift the cells, the medium was removed and cells were washed with 37°C DPBS. The DPBS was removed and replaced with 37°C 0.05% trypsin-EDTA (Lifetech #25200-056). Following a 3-5 minute incubation at 37°C, the plates were tapped to release cells and the enzymatic digestion was terminated with 3 volumes of media. Cells were pelleted at 200x g for 5 minutes at room temperature. Supernatant was discarded and 1 mL of media was used to suspend the cells. Cells were counted using the Nexcelom Auto2000 cellometer and the ViaStain AOPI staining reagent using the manufacturer's protocol and a built-in settings. At passage, the number of cells, percentage of live cells, cell size, and number of hours in culture were recorded. Cells were initially plated at a density of approximately 10,000 cells/cm²; using this as the initial cell number and the number of cells at harvest as the final cell number and culture time, population doubling time was calculated using the standard formula. At times, extra cells were frozen for later use. To freeze, cells were cryopreserved using a 1 : 1 ratio of HPL media and cryopreservative (Globalstem #GSM-4200), and held on ice until transfer to a controlled rate freezing device (Mr. Frosty) and being placed into a -80°C freezer overnight. The next day, the vials were moved to the vapor phase of liquid nitrogen for long term storage.

CFU-F assay. MSCs were plated at 10, 50 or 100 cells per cm² in duplicates in 6 well CytoOne tissue culture plates in 2, 5, and 10% FIPL enriched DMEM, as described above. Four cell lines were expanded 4 days in culture, prior to fixation and methylene blue staining. Subsequent tests used 4-7 days of culture at a density of 5, 10 or 50 cells per cm². After the required culture period, the medium was removed and the cells were washed with DPBS, then fixed using 4°C 100%> methanol for 5 minutes. The cells were washed again with DPBS, stained with 0.5%) methylene blue for 15 minutes, rinsed several times with distilled water, and air dried. The stained colonies were counted manually at 40x final magnification. Colonies were defined as isolated groups (clonal groups) of at least 10 cells. Colony number was determined by averaging the number of colonies in the technical replicates at each plating density for a given expansion period. Colony forming efficiency was calculated by dividing the number of plated cells by the number of colonies.

Differentiation. Differentiation of MSCs was induced by replacing the expansion medium with MSC differentiation medium (StemPro, Life Technologies #s Al 0070-01, Al 0071-01, Al 0072-01 for adipogenic, chondrogenic and osteogenic differentiation) and following the manufacturer's protocol. After about 21 days of differentiation, the differentiation medium was removed, the MSCs were washed with DPBS, fixed with 4% paraformaldehyde for 10 min, and stained with Oil Red for analysis of adipose cells, Safranin O for chondrogenic cells or Alizarin Red S for osteogenic cells. Micrographs were taken using an Evos FL Auto microscope (Life Technologies).

Flow Cytometry. The BD human MSCs flow cytometry characterization kit was used for positive and negative surface marker staining (#562245). Using the manufacturer's recommended protocol, MSC samples were stained with four fluorochromes together including positive and negative staining cocktails. The positive marker cocktail stained for CD90, CD 105, and CD73 (defined as > 97% positive staining). The negative cocktail (all antibodies were stained using a single fluorochrome, PE) stained for CD34, CD45, CD1 lb, CD 19, and HLA-DR (defined as < 2% positive staining). A CD44 labeled PE antibody was used as positive control for the negative cocktail to set the compensation and gating of the negative cocktail. For each flow cytometry run, fluorescence minus one controls for each fluorochrome and isotype controls for each antibody were used for compensation and non-specific fluorescence analysis. Samples were washed with 1% BSA solution before and after staining. A FACScaliber (BD Biosciences) was used for flow cytometry and analysis was conducted using FCS software. Negative staining gate of the isotype control was set at 1% positive staining.

Statistics. After confirmation that ANOVA assumptions of normality and homogeneity of variance were met, ANOVA was used to evaluate significant differences between variables. If the assumptions were violated, the data set was transformed mathematically and again tested to see if it met ANOVA assumptions. Hypothesis testing was two tailed (e.g., meanl≠ mean2). After running ANOVA and finding significant main effect(s) or interaction terms, post hoc means testing of planned comparisons was conducted using either the Bonferroni correction or Holm-Sidak method. Significance was set at p < 0.05. Data is presented as average (mean) plus/minus one standard error. In one case, in order to pass the normality test (Shapiro-Wilk) an "outlier" was removed. After the outlier was removed, the dataset passed the normality test and ANOVA determined that there was a significant effect of HPL concentration. SigmaPlot v.12.5 (Systat software) was used for statistics and making of the graphs. The graphs created in SigmaPlot were saved as EPS files and moved into a vector-based graphics package (Adobe InDesign or Adobe Illustrator CS6) for editing and rendering.

Results

Umbilical Cords. Umbilical cord from Caesarean-section delivery (n=17) and "normal" vaginal delivery (n=7) were used in this research. The biographic data of each cord is shown in Table 1.

Table 1 : Data from 24 umbilical cord MSC isolations.

uc- Gram

Length Weight

Sex Birth Enzyme per Viability ± SE

MSC (cm) (g) cm

241 F V 46 High 67.9 1.5 76.7% 0.6%

242 M V 43 High 60.7 1.4 58.0% 1.9%

243 F V 57 High 81.1 1.4 62.8% 2.0% 244 M C-S 35 High 66.0 1.9 58.6% 2.6%

245 M V 61 High 76.0 1.2 50.6% 6.4%

246 M C-S 41 High 60.9 1.5 66.0% 0.9%

248 F C-S 47 High 72.6 1.5 68.3% 3.2%

249 F C-S 32 High 37.7 1.2 64.2% 10.7%

250 M V 26 High 43.3 1.7 84.2% 0.5%

251 F V 28 High 30.4 1.1 74.4% 3.8%

252 M C-S 54 High 83.6 1.5 79.8% 3.9%

253 M V 61 low 48.8 0.8 58.0% 3.7%

254 F C-S 38 low 59.3 1.6 60.9% 3.9%

255 F C-S 47 low 82.3 1.8 75.1% 8.6%

256 M C-S 45 low 49.0 1.1 70.2% 1.1%

257 M C-S 43 low 105.2 2.4 66.6% 3.0%

258 M C-S 37 low 45.1 1.2 62.5% 4.6%

259 M C-S 31 low 57.8 1.9 62.2% 3.5%

260 F C-S 67 low 100.6 1.5 64.4% 1.8%

261 M C-S 51 low 92.8 1.8 72.3% 2.1%

262 F C-S 28.5 low 25.1 0.9 50.5% 4.1%

263 F C-S 28 low 23.8 0.9 65.3% 3.8%

264 F C-S 32 low 45.7 1.4 59.8% 3.4%

265 M C-S 38 low 38.7 1.0 54.8% 3.2%

UC- Live cells live cells per Theoretical

± SE ± SE

MSC per cm gram Cell Yield

241 3.7E+05 2.3E+05 3.2E+05 5.6E+04 2.17E+07

242 2.4E+05 2.9E+04 1.7E+05 2.2E+04 1.06E+07

243 3.9E+05 7.4E+04 2.7E+05 6.4E+04 2.17E+07

244 2.0E+05 2.4E+04 1.2E+05 1.8E+04 7.85E+06

245 3.3E+05 9.9E+04 2.7E+05 9.2E+04 2.06E+07

246 1.5E+05 4.3E+03 8.5E+04 8.2E+03 5.19E+06

248 2.9E+05 4.2E+04 1.2E+05 4.3E+04 8.66E+06

249 1.3E+05 4.9E+04 1.2E+05 4.3E+04 4.50E+06 250 5.3E+05 4.1E+04 3.1E+05 8.8E+03 1.34E+07

251 9.9E+04 5.7E+02 8.8E+04 1.7E+04 2.69E+06

252 1.9E+05 3.9E+04 1.3E+05 5.0E+04 1.11E+07

253 3.4E+05 9.8E+04 2.3E+05 6.5E+04 1.11E+07

254 1.1E+05 5.3E+04 7.9E+04 4.0E+04 4.68E+06

255 7.7E+04 5.2E+03 4.7E+04 4.0E+03 3.85E+06

256 2.4E+05 5.8E+04 2.1E+05 3.3E+04 1.02E+07

257 3.5E+05 2.4E+04 1.5E+05 1.4E+04 1.56E+07

258 1.9E+05 3.8E+04 1.6E+05 2.7E+04 7.01E+06

259 2.7E+05 2.6E+04 1.5E+05 2.2E+04 8.56E+06

260 1.7E+05 3.9E+04 1.1E+05 2.8E+04 1.11E+07

261 2.2E+05 1.9E+04 1.2E+05 8.8E+03 1.10E+07

262 1.0E+05 2.7E+04 1.5E+05 5.7E+04 3.81E+06

263 2.0E+05 5.4E+04 2.6E+05 8.9E+04 6.27E+06

264 4.5E+05 4.7E+04 3.2E+05 4.3E+04 1.46E+07

265 1.5E+05 3.0E+04 1.5E+05 3.5E+04 5.81E+06

24 cords were compared below

g per Live cells Live cells

Length Weight Viability Theo. Yield cm per cm per gram

Mean 42.4 60.6 1.4 65.25% 2.42E + 05 1.72E + 05 1.0LE + 07

Standard dev. 11.6 22.9 0.4 8.33% 1.16E + 05 7.48E + 04 5.00E + 06

Coefficiency

27.5% 37.7% 26.9 12.8% 47.9% 43.5% 49.7% of var.

F=Female, M=Male, C-S=Caesarian-Section and V= Vaginal. The enzyme concentration: low was 300 U/mL and high was 532 U/ml of Collagenase. SE=Standard Error, which was calculated after averaging the technical replicates for each umbilical cord. Live cells per gram was calculated from the live cell number for each tube divided by the weight of the tube. Theoretical yield calculation represents cell numbers achieved assuming the entire umbilical cord was processed and expanded.

Isolation Method Comparison. Note that the MSC isolation comparison was considered for passages 1-5, and passage 0 was considered part of the isolation of MSCs. Results obtained from a previous work by this lab (historical data from 27 umbilical cords) and the method described herein were compared. As shown in Fig. 2 A, the present method yielded on average 10 times more MSCs per cm length than the prior method and yielded MSCs in 100% of the UC samples. While bacterial, viral, or fungal contamination was not tested, no break in sterility was apparent here (e.g., no frank contamination was observed and no cultures were discarded due to contamination). Live cells per cm of length or per gram were compared in Table 1, there was a trend for the coefficient of variation to be less for live cells per gram. The present method used a closed processing system for tissue disruption and required a total of 4 hours of worktime including a 3 hour enzyme extraction step to isolate the umbilical cord MSCs. MSC attachment was observed within 24 hours of the isolation and proliferation was observed in all three HPL media enrichment conditions. As shown in Fig. 2C, during the isolation phase (pO) UC-MSCs grew more quickly when plated in 5% or 10% HPL enriched DMEM than UC-MSCs plated in 2% HPL enriched DMEM. It is possible that UC-MSCs grown in 5 or 10% HPL enriched DMEM attached more quickly than those grown in 2% HPL enriched DMEM in pO. The growth rate difference for 2% HPL enriched medium was statistically different (slower) at pO then later passages (see Fig. 2C and 3 A), and was significantly different (slower) than 5 and 10% HPL enriched media at isolation and during expansion.

MSC Expansion Comparison. Note that the MSC expansion comparison was considered for passages 1-5, and passage 0 was considered part of the isolation of MSCs. UC-MSCs were expanded for passages 1-5 here. UC-MSCs were evaluated in 3 different growth conditions: DMEM supplemented with 2% HPL, 5% HPL or 10% HPL. A two-way ANOVA (main effects HPL level and expansion over time) found a significant main effect (HPL concentration) on attachment and expansion. In post hoc testing, we found significantly more cells—about 30% more, were obtained when cells were expanded in 10% HPL enriched DMEM medium compared to 5% HPL enriched medium (9.4 x 10⁵ ± 6.2 x 10⁴ cells per cm² versus 6.6 x 10⁵ ± 3.8 x 10⁴ for 5% HPL enriched medium (Fig 3B). Similarly, post hoc testing showed significantly shorter population doubling times when MSCs were expanded in 10% HPL (32.4 ± 2.5 hours), compared to 40.7 ± 4.1 hours for 5% HPL and 100.9 ± 14.8 hours for 2% HPL enriched medium (shown in Fig. 3C). As shown in Fig. 3D, MSCs grown in 10% HPL enriched DMEM averaged 17% smaller than those grown in 2% HPL (14.7 ± 0.2 um vs 17.6 ± 0.4 um) and 10% smaller than cell grown in 5% HPL enriched medium (on average over 5 passages, 16.1 ± 0.3 um). The trends in MSC size across HPL medium conditions became noticeable after the second passage (Fig. 3E). HPL medium enrichment affected the viability of the cells noted at passage (see Fig. 3C). As mentioned in the Methods section, after we removed an "outlier", subtle but significant differences were found in viability at passage between the three medium conditions: MSCs in expanded in DMEM supplemented with 10% HPL had higher viability than those grown in DMEM supplemented with 2% HPL (92.2 ± 0.9% vs 84.9 ± 1.7%) and 5% HPL supplemented medium had significantly greater viability than 2% HPL medium (90.4 ± 0.9%; see Fig 3C).

The theoretical cell yield was calculated assuming the entire umbilical cord was isolated and expanded in each medium condition to passage 5. As shown in Fig. 3F, it was estimated that the total yield might exceed 10¹² MSCs (a trillion cells) at passage 5 for UC MSCs expanded in 10%) HPL supplemented medium and exceed 10¹¹ MSCs for UC MSCs expanded in medium supplemented with 5% HPL (Fig. 3F).

Evaluation of UCMSC characteristics. Sex of the donor had no effect on number of MSCs isolated (Fig. 2B), or the estimated number of MSCs obtained after expansion (data not shown). In contrast, a significant increase in the number of cells isolated was found for UC-MSCs isolated from normal vaginal delivery compared to those collected following Caesarian-section delivery (see Fig. 2B).

Colony Forming Unit - Fibroblast ( CFU-F) data is presented as a normalized unit: colony forming efficiency (CFE; CFE = number of plated cells divided by number of colonies). As shown in Fig. 4E, the concentration of HPL supplementation had no effect on CFE at 10 cells / cm² (100 cells per well of a 6 well plate) after 4 days of culture. In contrast, when plated at a density of 50 cells per cm² and 4 days of expansion in culture, 10%> HPL supplementation resulted in an increased colony forming efficiency compared to 2 and 5% HPL: 2-4 MSCs were needed to form a colony when plated in medium supplemented with 10%> HPL (Fig 4E). As seen in Fig. 4E, plating density affects colony forming efficiency and higher efficiency is found at lower plating density. Therefore, it was considered whether higher efficiency would be found at plating density below 10 cells/ cm² after plating in HPL. The highest colony forming efficiency was found when MSCs were plated at 5 cells / cm² for 6 days (50 cells per well of a six well plate); in medium supplemented with 10%> HPL: on average one out of two MSCs formed a colony (Fig. 6).

Differentiation. MSCs isolated and expanded using the method herein undergo differentiation to the three mesenchymal lineages, bone, cartilage, and fat, after exposure to differentiation medium conditions for 3 weeks. Figs. 4A-4D show MSCs differentiated to fat, chondrogenic, and osteogenic lineages following closed isolation method and expansion to passage 5 in 10%> HPL supplemented DMEM. Exposure to adipogenic differentiation medium resulted in formation of lipid droplets in MSCs that stained with oil red (Fig 4A). Exposure to osteogenic differentiation conditions resulted in calcium deposits formed within MSCs stained with Alizarin red S (Fig 4B). Cartilage-like tissue formation was observe in clusters of cells after exposure to differentiation medium as indicated by glycosaminoglycan staining by Safranin O for chondrogenic cells (Fig 4C).

Flow Cytometry. Flow cytometry was used to analyze the surface marker expression in 5 MSC lines following isolation using the closed processing protocol and expansion using the 10% FIPL supplemented DMEM for 5 passages. High expression (> 95% positive) for surface markers CD73, CD90, CD105 and CD44 was observed (Fig. 5 for representative results, Table 2 for all flow cytometry data). Low surface maker expression (< 0.5% positive) was observed for CD34, CD45, CD1 lb, CD 19, HLA-DRT (Table 2). To evaluate the effect of freezing and thawing MSCs on surface marker expression, four MSCs lines were evaluated before and after a freeze/thaw cycle. No significant differences were found in surface marker expression between frozen/thawed and never frozen MSCs in surface marker expression (Table 3).

Table 2

Table 3

Discussion In order to develop an SOP for GMP production of UC MSCs, limitations in the previous method were identified for UC-MSC isolation and expansion that represented barriers for GMP production. First, the previous isolation method required a lengthy dissection step, and the opening of the umbilical cord and manually removing the vessels prior to mincing the Wharton's jelly was time consuming and increased contamination risk. The method herein sought to reduce processing time and reduce contamination risks. A standardized method for liberating MSCs from the Wharton's jelly may produce a more homogenous product. Second, the previous UC-MSC medium was complicated, with more than 10 components, and it contained 2% FBS, a xenogeneic product. The method herein uses a simplified medium that is free of xenogeneic materials and contains fewer components. Human platelet lysate (HPL) enriched medium was tested. 5 or 10% HPL enrichment vastly improved MSC expansion in the pO (initial isolation). Furthermore, robust expansion was found over passages 1-5. Therefore, use of FIPL-enriched medium eliminated two barriers to GMP compliant manufacturing of UC -MSCs. However, using a pooled human blood product is not without certain risks, they have been somewhat mitigated (discussed below). Due to the sample to sample variability, pooling of platelet lysate is essential to produce a uniform product. For example, human pathogens that escape screening by the providers may contaminate HPL samples. The repeated freeze-thaw process followed by the filtration through a 0.2 um filter should remove all potential bacteria and parasites. While gamma irradiation is something that may be considered to lower viral risk, the blood products used were obtained from a blood bank for clinical use and thus had met all existing blood screening safety measures.

In this experiment, 1 cm sections of cord were used to optimize the protocol. The 1 cm length sections of umbilical cord provided enough cells for isolation and expansion, and many technical replicates were available from one cord, which allows multiple experimental variables to be examined in each cord (the biological variable). It was assumed that a randomly selected, one cm length of cord would adequately represent the umbilical cord (e.g., that cellular distribution and umbilical cord extracellular matrix is homogenous). Umbilical cords display tremendous biological variation in density (weight per unit length), diameter, and physical mechanical properties, perhaps due to the amount of extracellular matrix surrounding the vessels (see Table 1). The gram per cm measurements vary within each technical replicate from a single cord and between different umbilical cords, too. This indicates the importance for multiple biological and technical replicates when performing optimization testing using umbilical cords. It was assumed that the cells isolated after an initial passage are similar in physiology. Frank differences were not observed in the population of MSCs isolated following their extraction from Wharton's jelly versus those isolated following disruption of the entire 1 cm cord fragment. It was found that extraction of the entire 1 cm length using the methods outlined here gave a > 10 fold increase in the number of input cells for the primary culture over the prior methods. The reduced manipulation of the tissues (elaborate dissection negatively affects the attachment and expansion) and the more efficient removal of red blood cell contamination (blood negatively affects the viability, attachment and expansion of MSCs) are attributed to the improved extraction and expansion efficiency.

Previously, "cord length" measurement for comparisons of yield between cords was used. In this experiment, both length and weight were tracked to determine whether either proved to be a better predictor of cell yield in initial isolation. The variation between umbilical cords for both length and weight is represented in Table 1. As seen in Table 1, weight was a more reliable measurement compared to cord length.

The increase in cell numbers from the method herein may be attributed to the faster processing and reduced dissection when isolating the MSCs. By not removing the blood vessels, this method is a significant departure from previous methods.

This experiment further demonstrates a difference between MSC isolation efficiency from umbilical cords derived from vaginal births vs. Caesarian-section births. Vaginal birth umbilical cords had more cells per after isolation by approximately 41% (Fig. 2B). Prior to observation, Caesarian-section umbilical cords were preferred for MSC isolation because it was assumed that surgical collection would have a reduced contamination risk compared to cord collected following the passage through the birth canal. During this study, no differences were found in contamination from either vaginal birth or Caesarian-section umbilical cords. Similarly, the volume of umbilical cord blood collected was decreased by vaginal birth over Caesarian-section birth. No sex difference was observed between the number of cells isolated or MSC expansion rate or number. Enzymatic digestion using a high concentration of digestive enzymes tended to have higher yield at isolation (Fig. 2B). Visually, higher concentration of enzyme samples appeared to have less debris when compared at initial plating compared to lower enzyme concentration.

After the initial plating of cells during the isolation protocol, there is a delay in cell attachment. Attachment to the substrate is a defining characteristic of MSCs and appears to be necessary for MSCs expansion. After the isolation of MSCs, in passages 1-5, MSCs attach and begin to expand within 24 hours of plating. In contrast, the time to reach the confluence for the isolation and initial passage is significantly impacted by delays in attachment. In this experiment, the P0 data was included with the isolation of MSCs and passages 1 through 5 for the expansion phase of MSC characterization were considered. There was an observable trend that when there was a higher viability at the initial isolation, the cells attached better and expanded more rapidly. In prior work, cell viability was not recorded at the initial isolation. In this experiment, the use of the Nexcelom and ViaStain AOPI viability assay provided a quantitative method and gave more consistent results than trypan blue and manual counting using the haemocytometer (which is how we counted cells, previously). Automated cell counting lends itself to optimization and producing SOPs.

When considering the production of a public bank of cord samples, freezing the primary isolates at pO - pi is likely to be a necessity. Others have reported this affects cells surface marker expression or viability. For that reason, surface marker expression in never frozen cells and in cells subjected to a freeze/thaw cycle was evaluated. The flow cytometry analysis did not show a difference in surface marker expression between fresh cells (e.g., those never frozen) and cells frozen and thawed cells for four umbilical cord MSCs lines. Clinical trials will require the freezing of cells for use, the use of fresh cells in clinical trials is not feasible when considering the rigorous quality control and release testing that must be done to determine if these cells meet the standards for clinical use.

In this experiment, human platelet lysate enriched media at three different concentrations (2%, 5%, and 10%) was used to analyze the effect on the initial isolation (pO) and growth of the MSCs for passages 1-5. MSCs were evaluated through passage 5 to characterize expansion potential. It was observed that PO to PI expansion exhibited the highest amount of variation in growth rate (see Figs. 2C and 3A). Results from six umbilical cords did not show a difference between passages 1-5 for population doubling time, number of cells at passage and viability at passage (data not shown). However, the three media conditions did affect these variables. Enrichment with 2% HPL enriched medium was significantly different than 5% and 10% HPL enriched media for population doubling, cell size, cell numbers and viability. Enrichment with 2% HPL had slower population doubling, fewer cells at passage, larger cells and a lower percentage of viable cells at passage. A trend was observed associated with better results for these measurements as HPL enrichment in the medium increased. For this reason, 10% was chosen as the new standard media condition to be used to grow the UC-MSCs. Unexpectedly, cell size for the UC-MSCs was a variable that varied significantly between the different media conditions. Also surprisingly, there was a trend for cell size to initially increase after the first passage then decrease over subsequent passages in all media conditions (Figs. 3D-E).

Validation that the cells isolated and expanded were MSCs was done by assessing cell surface markers with flow cytometry, CFU-F and differentiation capacity. All five cell lines analyzed with flow cytometry had high levels of the surface markers known to be associated with MSCs. The high percentage of positive cells (>95%) is comparable to the previously published method. These results suggest a homogenous cell population was isolated even though the blood vessels were not removed for the isolation step. Differentiation ability was assessed in the same five cell lines and all display trilineage differentiation capacity. The capacity for adipogenic differentiation was analyzed by Oil Red O staining for lipid droplet accumulation within the differentiated cells cytoplasm. Analysis showed multiple lipid droplets forming within a large number of the cells (Fig. 4A). Cell death did occur during the time to differentiate, leading to space between the adipogenic cells in the figure. Osteogenic differentiation was stained with Alizarin Red S to analyze calcium deposit formation. Staining was observed in calcium deposits on the cells and within the cells as seen in Fig. 4B. Chondrogenic differentiated cells were stained with Safranin O to assess if cartilaginous associated with glycosaminoglycan. This differentiation yielded circular colonies, often remaining adhered to the plate and they robustly stained for Safranin O. Typically histological sections of microcolonies are used to assess chondrogenic lineage differentiation. The results indicate this is not necessary when small colonies of cell remain adherent (Fig. 4C). Although the results are not quantified, the quality of the staining and duplication between multiple lines provides good evidence that MSCs isolated and expanded by the method described herein have robust trilineage differentiation potential.

Colony forming unit fibroblast (CFU-F) efficiency analyzed self-renewal potential of UC- MSCs. Compared to previous research for UC MSCs expanded in 21% oxygen, fewer cells were needed to form a colony using the method described herein, suggesting a higher colony forming efficiency. The 10 cells per cm² were considered a more reliable measure for the effect of FIPL concentration due to difficulty counting cells at 50 cells per cm². The fast growth rate for the 10% FIPL enriched medium made the previous CFU-F protocols unreliable because the plates grew too fast. Growth conditions for measuring colony forming efficiency were tested by analyzing days from plating vs. colony counts. It was found that the number of cells to form colonies decreased with each day of growth, the exception was 50 cells per cm², which increased. The highest CFU- F efficiency was for 5 cells per cm²cells grown for 6 days. It was determined that using both 10 and 50 cells per cm² yield consistent data for CFU-F. The self-renewal data (colony forming efficiency) is important when estimating the expansion potential of a MSC line. Higher CFU-F efficiencies are associated with MSC lines displaying a more robust growth potential. Determining the method for analyzing CFU-Fs in these fast growing cells allows for analysis of growth potential for future research using UC-MSCs.

We provided herein an improved method to isolate and expand UC-MSCs. When compared to our previous method, an increase in total MSC yield at the initial isolation of more than 10 times was obtained, and less time is needed to isolate MSCs from the umbilical cord. Additionally, this method reduces the overall expansion by reducing the number of population doubling needed to meet our production target of 2-10 billion cells per batch. The method uses closed system for initial isolation with minimal dissection of the cord, and, thus, reduces contamination risk, while simultaneously reducing processing time. The method uses a simplified (5 component), xenogen-free medium that can be upgraded to GMP-compliant components for scale up. Characterization of MSCs produced using this improved processing protocol and simplified medium included in vitro expansion, colony forming efficiency, trilineage differentiation to osteogenic, chondrogenic and adipogenic lineages, and surface marker expression by flow cytometry indicate that MSCs were produced by this method.

Conclusion

The methods developed for isolation and expansion of UC-MSCs address some challenges to translation to clinical use. An increased MSC yield for vaginal births compared to Caesarian section births was reported. The new isolation method provides the necessary cell yield for banking and uses a closed system that can be easily scaled up and expansion media supplemented with 10% FIPL had the best growth rate. These results provide improvements that may support GMP manufacturing of UC-MSCs.

EXAMPLE II

The great properties of human mesenchymal stromal cells (hMSCs) make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during the isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, this experiment investigated a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cell growth in a 12-well-plate and a spinner flask. The goal was to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor.

A microcarrier-based process was considered for the expansion of hUC MSCs in a high surface-to-volume ratio. The goal was a robust and reproducible process according to GMP and GCP. To achieve this goal, this study focused on the characterization of the cell growth and metabolism of hUC MSCs and the process transfer from static culturing to a dynamic system. A small-scale spinner flask culture was used to identify the culture conditions for the expansion in the bioreactor. The crucial culture conditions we evaluated were the inoculation strategy, seeding density, stirrer speed, microcarrier type, and batch or fed-batch cultivation. We strived for a cultivation strategy with a high hUC MSC harvest yield while achieving all quality control criteria of these cells.

Materials and Methods

Umbilical Cord and Cell Isolation. Five human umbilical cords were used for this work. The cords were discarded tissues from apparently healthy, anonymous donors. The MSCs were isolated from the umbilical cords using the method described above. For this study, cells at passage 3 were used.

Culture Medium. The culture medium for hUC MSCs was based on DMEM low glucose (LG) medium (Gibco^® by Thermo Fisher Scientific, Cat. No. : 11885-084) with addition of 10% pooled human platelet lysate (hPL, pooled from more than 25 expired units), 1% GlutaMAX™ (Gibco^®, Cat. No.: 35050-061), 1% Antibiotic-Antimycotic (Gibco^®, Cat. No.: 15240-062) and 0.4% Heparin (1000 USP U mL^"1). All components were combined and sterile filtered (0.22 μπι, Corning^®, Cat. No.: 431098). The high glucose medium was made by the same procedure, but the DMEM LG medium is replaced by DMEM high glucose medium (Gibco^®, Cat. No. : 11995-065).

Growth Kinetic under Static Culture Conditions. The growth kinetic of five cell populations from different cords (two male donors, three female donors) in 2D was investigated in a 12-well-plate (CytoOne, USA Scientific, Item No. : CC7682-7512) to determine the growth parameter and as a comparison to the cell growth in the dynamic system. The cells (passage 3) were seeded at a density of 8000 cells cm^"2 and a working volume of 1 mL DMEM LG medium. The cells were cultured for 7 days and harvested. To harvest, the medium of three wells was aspirated, washed once with 1 mL Dulbecco's phosphate buffered saline (DPBS), and lifted from the substrate with 0.05% trypsin-EDTA (Gibco^®, Cat. No. : 25200-056). The cell number, cell viability and cell size were determined with the Cellometer Auto 2000 Cell Viability Counter using Viastain AOPI staining kit (both from Nexcelom Bioscience, Waltham, MA).

Microcarrier Selection. For the expansion of hUC MSCs on microcarner, five different microcarrier types (Animal Product Free Starter Kit, SoloHill^® by Pall Corporation) were compared (three-fold determination, n=3) to achieve the best growth and attachment conditions for the hUC MSCs. The starter kit contains Hillex^® II, Plastic Plus, Plastic, ProNectin F-coated and Glass-coated microcamers (Table 4). In addition to these five microcarrier types, the effect of hydrogen chloride treatment on the Glass-coated microcamers was tested. The Glass-coated microcamers (100 g) were incubated in a bottle with 100 mL 1 M HC1 over night (16 h) at room temperature. After the incubation, the microcamers were washed and dried. For each microcarrier type, 1 g microcamers were autoclaved with 3 mL DPBS at 121°C and 1 bar over pressure for 20 min. The microcarrier selection was performed in an ultra-low attachment 6-well-plate (Corning^® Costar^®, Sigma-Aldrich^®, Cat. No.: CLS3471-24EA) with a seeding density of 8000 cells-cm^"2, 1.5 mL DMEM LG medium, 25 g L^'1 microcamers over 3 days for each microcarrier type. Every day, the cells of three wells were harvested. 1 mL was taken into a reagent cup, the microcamers settled to the bottom, supernatant removed, added 400 μΕ of trypsin-EDTA (0.05%), and incubated the sample for 5 min under swirled conditions at 37°C and 5% CO2. The reaction was stopped with 800 μΕ of hPL containing fresh and pre warmed culture medium, and the cells were separated from the microcarrier through a cell strainer (Miltenyi Biotec, MACS SmartStrainers, 30 μπι, Cat. No. : 12-565-271). The cell strainer was rinsed three times with 1 mL DPBS. After centrifugation (5 min, 200 x g), the supernatant was discarded, and the cells were re-suspended in fresh and pre warmed culture medium. Cell number, cell viability, and cell size was determined with the Cellometer Auto 2000 Cell Viability Counter.

Table 4: Microcarrier specification for Animal Product Free Starter Kit by SoloHill®, Pall Corporation

Spinner Cultivation. The hUC MSCs were cultured in spinner flasks (Bellco, IOB W/ MC FLASK, 100 mL, Cat. No.: 1965-61001) with 100 mL working volume and 25 g-L^"1 microcarriers. The cells were seeded at 8000 cells- cm^"2 and DMEM LG medium (see 0). For the first 24 hours, the cells were cultured with 25 rpm stirrer speed to facilitate the attachment of the cells. After 24 hours, the stirrer speed was increased to 40 rpm and every second day, the speed was increased about 10 rpm to avoid microcarrier agglomeration. A 50% medium exchange was performed when the glucose concentration in the medium dropped to 0.2 g-L^"1. The medium was exchanged with fresh and pre warmed culture medium with a glucose concentration of 2 g-L^"1 to achieve an end concentration of 1.2 g L^'1 in the spinner flask. Every day a sample was taken from the spinner flask to determine cell number, cell viability, cell size, and metabolites (see 0). After 6-8 days of culture, the entire spinner was harvested. To harvest, the stirrer speed was set to 0 rpm to let the microcarriers settle down and as much as possible (80-90 mL) of the culture medium was removed. The microcarriers were washed two times with 20 mL pre warmed DPBS and incubated with 15 mL pre warmed 0.05% trypsin for 10 minutes at 37°C and 5% CO2 to detach the cells from the microcarriers. The trypsin reaction was stopped with 40 mL fresh and pre warmed culture medium. The cells were separated from the microcarriers through a cell strainer (30 μηι) and rinsed with DPBS. After centrifugation (5 min, 200 x g), the supernatant was discarded and the cells were re- suspended in fresh and pre warmed medium to determine cell number, cell viability and cell size.

Differentiation Analysis. After spinner harvest, hUC MSCs were seeded in a 12-well-plate (CytoOne, USA Scientific, Item No. : CC7682-7512) to confirm the differentiation capacity. This was performed according to the protocols of the StemPro^® differentiation kits for adipogenesis, chondrogenesis and osteogenesis by Thermo Fisher Scientific.

Cell Count and Maximal Growth Rate. Cell count, cell viability, and cell size were determined with the Cellometer Auto 2000 Cell Viability Counter using the Viastein AOPI staining kit (both from Nexcelom Bioscience, Waltham, MA, USA). The exponential growth phase of the cells was estimated from a line created by plotting the natural logarithm of the cell number against the culture time. The slope of this linear curve is determined by linear regression analysis (GraphPad Prism, Version 5.01, GraphPad Software Inc.), which represents the maximal growth rate.

Microscopic Analysis of Cell Growth. The qualitative analysis was done by staining the cell nuclei on the microcarriers with SYBRGreen I (10,000x concentrate in DMSO, Cat. No.: S- 7563). The stock solution of SYBRGreen I was diluted 1 : 10000 with DPBS for staining. For detection with a fluorescent microscope (EVOS® FL Auto Imaging System), an absorption wavelength of 497 nm and emission wavelength of 520 nm was used. The distribution and occupancy of microcarrier with cells was determined by counting the number of microcarriers with and without cells.

Metabolite Analyzation. The metabolites glucose, glutamine, lactate and ammonium were measured in the cell culture medium. The metabolite concentrations were measured using a BioProfile^® 400 (Nova Biomedical, Waltham, MA). The consumption and production rates were determined by plotting the metabolite concentration over the culture time and calculation of the slope for linear consumption or production using linear regression analysis. The ratio of produced lactate to the consumed glucose called lactate yield and the ratio of produced ammonium to consumed glutamine (ammonium yield) for the static and dynamic cultivation were determined between 24 and 48 h. These yields serve for classification of the metabolism pathway.

Flow cytometry. The flow cytometry methods employed here are identical to those described in the previous example to permit comparisons between the results of MSCs expanded in static culture with those expanded in dynamic culture. Briefly, the BD Stemflow™ Human MSC Analysis Kit (BD Biosciences, Cat. No. 562245) was used for positive and negative surface marker staining. Using the manufacturer's protocol, hUC MSC samples were stained with four fluorochromes together including positive and negative staining cocktails. The positive marker cocktail stained for CD90, CD 105, and CD73 (defined as > 97% positively stained cells). The negative cocktail (all antibodies were stained using a single fluorochrome, PE) stained for CD34, CD45, CDl lb, CD19, and HLA-DR (defined as < 2% positively stained cells). A CD44 labeled PE antibody was used as positive control for the negative cocktail to set the compensation and gating of the negative cocktail. For additional methodological details, see the description in the previous example.

Colony-forming Unit Fibroblast Assay. The CFU-F methods employed here are identical to those described in the previous example to permit comparisons between the results of hUC MSCs expanded in static culture with those expanded in dynamic culture. Briefly, hUC MSCs were plated at 10, 50 or 100 cells-cm^"2 in duplicates in 6-well CytoOne tissue culture plates in DMEM LG. Cells were expanded 4 days in culture, prior to fixation and methylene blue staining. For additional methodological details, see the previous example.

Karyotype analysis. Following expansion of hUC MSCs in dynamic culture, the cells were cryopreserved to mimic how the cells might be banked prior to therapeutic application. The cells were subsequently thawed and plated at 1 - 1.5 x 10⁴ cells-cm^"2 in DMEM LG, as described in the previous example. Once attachment and expansion of the hUC MSCs were confirmed, the tissue culture flask were submitted to Cell Line Genetics for karyotype analysis (Cell Line Genetics, Madison, WI).

Results

hUCMSC Growth Kinetic under Static Culture Conditions. The growth kinetic in 12-well- plates was done to identify differences in the cell growth and metabolism of hUC MSCs from five donors (Table 5) and serves as comparison for the dynamic system. Fig. 7 shows representative cell growth data of hUC MSCs from one male and one female donor. All cells were in passage number 3 for better comparability and to avoid any influence of senescence. No significant difference was observed between the cells from female or male donors. The exponential growth phase for all five cell types was observed between 20 and 100 h of culture time. The highest cell concentration at the end of the exponential growth phase reached values from 1.3 x 10⁵ to 2.1 x 10⁵ cells-cm^"2 The mean value for the maximal growth rate μηωχ was 0.042 ± 0.005 h^"1 with a resulting doubling time of 16.8 ± 1.9 h and a mean fold expansion of 11.4 ± 0.7 after 7 days. Table 5: Maximal growth rate, minimum doubling time and fold expansion of hUC MSCs from five donors, after 7 days in culture in 1 mL DMEM LG medium in a 12-well-plate at a seeding density of 8000 cells-cm^-2 (n=3).

Max. growth rate Min. doubling time

HUC # Gender Fold expansion

255 female 0.045 ± 0.002 15.4 ± 0.6 12.7

260 female 0.049 ± 0.002 18.1 ± 1.3 11.0

262 female 0.037 ± 0.004 17.3 ± 1.7 10.9

256 male 0.038 ± 0.003 14.2 ± 0.5 11.1

257 male 0.040 ± 0.004 18.8 ± 2.1 11.2

Average 0.042 16.8 11.4

Standard Deviation 0.005 1.9 0.7

The glucose-lactate (A) and glutamine-ammonium (B) profiles of hUC MSCs from a female and male donor are shown in Figs. 8A-8B. After 3 days the concentration of glucose in the medium was under the detection limit of 1.1 mmol L^"1 and the lactate concentration reached a constant level of 4.5 mmol L^"1 after 4 days. The glucose-lactate profile in relation to the cell growth (Fig. 7) determined the glucose as limiting substrate for the hUC MSC proliferation. It was determined for hUC MSCs from a male donor a YLAC/GLC of 2.6 and for a female donor of 2.1. The consumption of glutamine was not limited and is connected with a constant production of ammonium. The ammonium yield YNH4/GLN of hUC MSCs from a male donor was 0.8 and for a female donor 0.5. A difference in the metabolism of hUC MSCs from male and female donor was not identified.

Microcarrier Selection. The microcarriers play an important role for the expansion of adherent cells. For this reason, a microcarrier selection between five microcarrier types was done (Fig. 9). The highest confluence and occupancy were the two crucial criteria for the selection of a suitable microcarrier type. The Pronectin F, Plastic and Plastic Plus microcarriers showed a similar occupancy between 73-75%. The obtained cell number from the Pronectin F and Plastic was lower than from the Plastic Plus microcarriers. Glass-coated microcarriers were investigated as untreated and pretreated with HC1. The pretreatment had a positive effect, which was visible in the higher final cell number. However, both Glass-coated microcarrier types showed a low microcarrier occupancy of around 50% (Fig. 10). In consideration of the confluence and the occupancy, the Plastic Plus microcarriers were the best choice for the expansion of hUC MSCs.

hUC MSC Expansion Using Dynamic Spinner Cultivation. Spinner flasks are a useful instrument for small-scale investigation of dynamic cell expansion. Inoculation methods with different resting times up to 4 h and occasionally briefly agitations were tested. There was no increase in cell growth or cell distribution based upon five independent tests (data not shown). We observed that inoculation strategies using agitation-rest-cycles led to an unwanted microcarrier agglomeration. Therefore we decided for an inoculation strategy under low (25 rpm) but continuous stirring. A seeding density of 8000 cells- cm^"2 was found as suitable to avoid large pre- cultures for inoculation and to prevent long culture times. To prevent microcarrier agglomeration with increasing cell number, the stirrer speed was increased every second day. The Plastic Plus and HCl-treated Glass-coated microcarriers were tested in nine spinner flasks trials. A non- homogeneous cell distribution on the HCl-treated Glass-coated microcarriers was observed in the spinner flasks. Also the cell yield for HCl-treated Glass-coated microcarriers was lower compared to the Plastic Plus microcarriers (data not shown). We observed no difference in the growth between cells from male and female donors on Plastic Plus microcarriers (Table 6, Fig. 11). In comparison to the static culture, the determined growth rates from the spinner flasks were comparable. The hUC MSCs could be efficiently detached with trypsin. An average cell yield of 4.2 x 10⁷ ± 1.4 x 10⁷ cells from an entire 100 mL spinner is related to a desired cell confluence of about 4.6 x 10⁴ cells-cm^"2 This high efficiency of harvesting hUC MSCs was confirmed by observing bare microcarriers after SybrGreen staining of the microcarriers following cell harvest. Table 6: Growth characteristics for hUC MSCs on Plastic Plus microcarriers in spinner flasks with 100 mL DMEM LG culture medium at a seeding density of 8000 cells-cm^"2

Culture Fold Highest Harvested

Gender ling time

time [d] expansion occupancy [%] cell yield

male 7 0.046 ± 0.006 14.9 ± 2.2 16.4 81 2.6 x 10⁷ female 6 0.032 ± 0.003 21.6 ± 2.5 13.8 93 5.3 x 10⁷

With glucose identified as the limiting substrate, a 50% medium exchange was done before the glucose concentration dropped under 1.1 mmol L^"1. Figs. 12A-12B show a representative metabolite profile of hUC MSCs from a male donor in a spinner flask culture. The medium was exchanged after three and five days to achieve a desired glucose concentration of 5.5 mmol L^"1. We determined a YLAC/GLC of 1.8 and YNH4/GLN of 0.7 between 24 and 48 h expansion time. The ground metabolism of hUC MSCs in spinner flasks cultures showed no difference between cells from a male and female donor.

Bead-to-Bead Transfer. The bead-to-bead transfer was carried out with a feed of fresh medium and microcarners after the first microcarriers were confluent. The working volume and microcarrier mass were doubled. The cells were cultured up to ten days and a 50% medium exchange was performed, when the glucose concentration in the medium dropped under 1.1 mmol L^"1. As criterion for the bead-to-bead transfer, the cell distribution on the microcarriers was analyzed. After two to three days a slight increase of the occupancy to a constant level about 60- 70% was obtained. In contrast to the rapid rise of the occupancy over 80% after inoculation, the cells did not appear to migrate to fresh microcarriers. At the end of the culture period, the microcarriers were either fully confluent with cells or completely devoid of cells (blank) (Fig. 13).

Quality Control of the hUC MSCs. After the harvest of the spinner flaks, the cells were seeded in 12-well-plates to prove their ability to differentiate to adipocytes, chondrocytes and osteoblasts. The dynamic expanded hUC MSCs were positive for lipid vesicle-forming adipocytes (Fig. 14, left), for calcium deposit producing osteoblasts (middle), and nodule forming chondrocytes (right). Further, it was proven that the growth characteristics did not change after a spinner cultivation. We determined no change in the cell growth and a high cell viability over 90%. Flow cytometry analysis revealed that the 3D cultured hUC MSCs were positively stained for surface markers of MSCs and were negative for markers of the hematopoietic lineage (see Figs. 15A-15E). CFU-F analysis revealed that dynamically cultured MSCs had colony forming efficiency (CFE, defined as number of MSCs plated divided by the number of colony forming unit - fibroblast) of between 4.5 and 2.5 (see Fig. 16). Finally, after reviving hUC MSCs that had been previously expanded in dynamic culture, we found that 90% of the cells were viable at thaw. These cells were plated in static culture in 10% FIPL enriched DMEM and they expanded robustly. The hUC MSC were sent for karyotype analysis at passage 5, and the karyotype was normal (see Fig. 17). These results indicate that dynamic produced hUC MSCs meet ISCT MSC minimal definition. Discussion

The goal was to investigate variables associated with the manufacture of hUC MSCs in a xeno-free, scalable, dynamic culture system. Three novel observations were made. First, after testing five different microcarriers with differing topography and surface chemistry, we found that Plastic Plus microcarriers were optimal for hUC MSC expansion. Second, after measuring medium metabolites during expansion, we found that glucose concentration was the critical variable for maintaining exponential MSC expansion. Third, by observing microcarrier occupancy, we determined that bead to bead transfer of MSCs does not occur to a significant degree. This is important for designing scalable expansion end points.

Using the International Society of Cellular Therapy minimal definition, the hUC MSCs grown in static culture and in the xeno-free, dynamic cultivation met the minimal MSC definition; this observation is in agreement with previous work of microcarrier-based expansion of MSCs or more specifically hUC MSC. Moreover, the colony forming efficiency of dynamically culture hUC MSCs is comparable to that found in static culture. Finally, a normal karyotype was found of hUC MSCs after microcarrier-based dynamic expansion, cryobanking, and expansion in static culture. These findings provide a basis for more highly refined microcarrier-based bioreactor experiments, scale-up, and validation studies. These results, together with the hUC MSC isolation and xeno- free expansion work described above, address an important information gap clinical manufacturing needs.

Comparison ofhUCMSCs Expanded in Static and Dynamic Cultivation. In comparison to

BM MSC, the proliferation rate of hUC MSCs is higher, which results in lower doubling time. The determined max. growth rate of 0.042 ± 0.005 h^"1 (to= 16.8 ± 1.9 h) is ~2-fold higher compared to the reported growth rates in BM MSC literature. A population doubling time has been determined of 27.5 ± 0.2 h for BM MSCs, which corresponds to a max. growth rate of 0.025 h^"1, and a growth rate has been reported of 0.020 ± 0.004 h^"1 (to=35.5 ± 6.00 h) for BM MSCs. The higher proliferation rate of hUC MSCs could be one explanation for the difference in the growth rates. The influence of the glucose concentration in the medium on the cell growth has been reported. They used an immortalized cell line hMSC-TERT and reported a max. growth rate of 0.039 h^"1 for the cells in low glucose medium (EMEM, 1 g L^"1 glucose) and a max. growth rate of 0.03 h^"1 for the cells in high glucose medium (DMEM, 4.5 g L^'1 glucose). This shows the influence of the medium and the components on the MSC growth. In regard that every research group may use different medium formulations makes it difficult to compare the cell growth between different laboratories. For this reason, the growth kinetic was performed under static culture conditions to obtain the standard and as comparison for the dynamic system.

The challenge of the process transfer from static to dynamic system is the differences of the growth surfaces and the connected process of cell adhesion (static flat bottom to suspended round microcarrier). The higher cell density with sufficient nutrient supply, and the potential harmful effect of shear forces created by stirring or aeration both may impact the proliferation of sensitive cells. The growth rate can be used as criterion to compare the different systems. The max. growth rate of 0.038 ± 0.008 h^"1 in the spinner flask is comparable to the max. growth rate of 0.042 ± 0.005 h^"1 from the 12-well-plate and both systems show similar growth kinetics (Fig. 7, Fig. 11). Additionally, in both systems no influence of the gender from the donor observed. The final cell confluence of 5.9 x 10⁴ cells-cm^"2 for the cultivation of the cells from a female donor is comparable with the reported concentrations from other studies.

Glucose is the primary source for mammalian cells to generate ATP by oxidative phosphorylation or by anaerobic glycolysis. The YLac/Gic indicates which metabolism pathway the cells preferable use to produce energy. For the hUC MSCs in static culture, a YLac/Gic of 2.6 (male) and YLac/Gic = 2.1 (female) was obtained, and for the dynamic system a YLac/Gic = 1.8 (male). It has been stated in literature that a YL₃C/GIC of 2 indicates that the cells use the inefficient glycolytic pathway instead of the oxidative phosphorylation to generate energy. The decrease of the YLac/Gic from static to dynamic hUC MSC expansion could be explained by changing the cellular metabolism from the glycolytic pathway to the oxidative phosphorylation by higher oxygen transfer in the spinner flask. The agitation in the spinner flask provides a homogenous distribution of substrates like glucose or oxygen in the culture medium, while the mass transfer in the static culture is driven by diffusion resulting in nutrient gradients.

Glutamine is the second main substrate for mammalian cell cultures and gets metabolically deaminated to glutamate. The hUC MSCs were evaluated for a YNH4/Gin of 0.8 (male) and a YNH4/Gin of 0.5 (female) in the 12-well-plates and a YNH4/Gin of 0.7 (male) for the dynamic culture. Other studies have shown YNH4/Gin of 1.6, but they provided reasons for not using the glutamine consumption and Fb production for characterization: Glutamine in the medium decays spontaneously and Fb gets formed spontaneously. Assuming about ± 10% glutamine decomposition per day, those studies calculated a glutamine consumption close to zero. Here, 2 mmol glutamax (glutamine dipeptide) was used, which is a stabilized form of glutamine, and 4 mmol glutamine in the culture medium. Fb, besides being formed from glutamine decomposition, is also formed in the metabolism of several amino acids and the Fb decreases by evaporation from the cultivation medium. These factors make it challenging to generate an accurate result about the glutamine metabolism of the cells. However, the daily 10% thermal glutamine decay in the static culture was calculated and shown in Fig. 8. The difference in the slope of the thermal decay and the curve of glutamine consumption by the hUC MSCs showed that the cells consumed the glutamine in the medium and the glutamine decrease cannot be reduced on the thermal decay. The glutamine consumption of hUC MSCs needs further investigations and for this reason, the supply of the glutamine should be given only by glutamax to differentiate between glutamine consumption by the cells and glutamine decay.

With increasing concentrations of lactate and ammonia, cell growth can be affected by a change of the pH and the cell toxic properties of these molecules. Studies have evaluated the toxic concentrations of lactate and ammonia for BM MSCs and found that cell growth decreased at a lactate concentration of 35.4 mmol and an ammonia concentration of 2.4 mM. During the cultivation of the hUC MSCs in the 12-well-plates these concentrations of lactate were never reached. After ± 120 h the toxic level of ammonia were exceeded in the 12-well-plate, which could cause an increased cell death. On the other hand, this experiment did not determine the toxicity of lactate or ammonia in hUC MSCs herein, and it has not been reported in the literature. Due to the 50% medium exchange in the spinner flask, it is likely that toxic levels of lactate and ammonium were not reached.

Five different hUC MSC isolates (3 female and 2 male) were expanded in static and dynamic cultivation here. In the present study, no frank differences were observed between MSCs expanded in static or dynamic culture in differentiation capacity using the described qualitative differentiation assays. In contrast, other studies that expanded hUC-MSCs in static culture or dynamic culture using 10% fetal calf serum enriched medium, reported subtle differences in surface marker expression (static culture hUC MSCs produced with positive CD349 staining), gene expression and cytokine secretion, but did not report difference in differentiation potential between static and dynamically culture. Other studies have suggested that cultivation of MSCs on microcarriers may impact osteogenic differentiation potential, perhaps due to the shear stress that MSCs grown on microcarriers are subject.

Microcarrier Selection and Bead-to-Bead Transfer. Cells derived from vertebrates have a heterogeneous negative charge on their surface. Suitable surfaces for the cell adhesion are dextran, glass, or plastic whose surface can be modified. During the adhesion process, electrostatic forces and van-der-Waals forces play an important role for the interaction of the cell and the growth surface. Divalent cations and glycoproteins from the medium are crucial factors for cell adhesion. These factors show the importance of a suitable growth surface and influence cell proliferation. The higher occupancy of the Pronectin F, Plastic and Plastic Plus microcarriers compared to the Glass-coated microcarriers (Fig. 10) leads to the preliminary conclusion that an uneven microcamer surface such as the cross-linked polystyrenes surface of these microcarriers provides more homogenous cell distribution and/ or better cell attachment. The positive effect of the hydrogen chloride treatment on the Glass-coated microcarrier could occur through a modification of the microcarrier surface. The microcarrier surface may get charged or rougher (etched) like the modified cross-linked polystyrene surfaces of the other microcarriers, which could help the cells to attach. The acid may also remove the glass surface and exposes the plastic core of the microcarrier. These results support the conclusion that hUC MSCs prefer plastic as substrate for attachment and confirms that the Plastic Plus microcarrier were a good choice.

The bead-to-bead transfer was based on the assumption of cell detachment from the growth surface and re-attachment following cell division. One reason for unsuccessful bead-to-bead transfer could be the ability of the cells to re-attach on the microcarriers in a stirred system, but the cells showed this ability during the dynamic inoculation period. With this background the basic assumption of detachment and re-attachment could be wrong, which covers the observation of certain other studies. For this reason, a homogenous distribution of the cells during the inoculation period is important. However, other studies have shown a successful bead-to-bead transfer with BM MSCs and collagen-coated microcarriers (SoloHill), which indicates the microcarrier as another source of error. This experiment focused the microcarrier selection on the goal of a high cell yield and not a good bead-to-bead transfer.

Influence of Donor and Source on Cell Proliferation. In this experiment, the gender of the cell donor had no influence on the cell growth and metabolism. The mentioned difference in cell proliferation of MSCs derived from the bone marrow and umbilical cord could result from the relative age of the cells. MSCs derived from the umbilical cord are in fact fetal cells and compared to bone marrow MSCs, which are usually isolated from adults. Also, it is generally thought that stem cells with a low age have longer telomeres and have the capacity for extended expansion in culture and reduced senescent. This experiment did not evaluate whether hUC MSCs grown in dynamic culture maintain long telomeres. Previous work described the long telomere length and telomerase expression of statically cultivated hUC MSC. Flow cytometry determined that dynamically expanded hUC MSCs did not have different surface marker expression for the ISCT positive and negative surface marker set (see Fig. 15). This finding is in agreement with other studies that found similar surface marker expression for UC MSCs for the ISCT marker set but different expression in CD349 (differentially expressed in statically cultured hUC MSCs and also adipose derived MSCs, but not those expanded in the bioreactor on microcarriers). Colony forming efficiency (CFE) of dynamically expanded UC MSCs was 4.5 - 2.5, which falls within the range observed for hUC MSCs expanded in static culture. This indicates that UC MSCs maintain a high degree of self-renewal capacity following bioreactor based expansion, and suggests that hUC MSCs remain stemmy or 'young' . Finally, the karyotype of hUC MSCs expanded in dynamic culture and frozen and thawed was found to be normal. This indicates MSCs subjected to the higher cytoskeletal stress (e.g., shear stress) of dynamic culture and freeze/ thaw stress were genetically stable. Therefore, the procedures used to hUC MSC expansion may be safe for use in cellular therapy.

There is no limit in the age to donate BM MSCs, which is why the age of the cells from donor to donor can change strongly with range of many years. In addition, the bone marrow contents change over the lifespan. The red marrow space changes to a yellow marrow by fat deposition, which complicates the extraction. Another aspect on the quality of the isolated cells could be the donor and the donor^'s physical health. The kind of lifestyle (healthy, unhealthy) or if the donor has/ had any diseases could affect the condition on the cells. These aspects may have more impact on the cell proliferation and expansion than the gender of the donor.

Conclusion

This experiment identified the relevant process parameters for a microcarrier-based expansion of hUC MSCs. To prevent microcarrier agglomeration a dynamic inoculation strategy and an increase of the agitation over the culture time is required. A microcarrier concentration of 25 g ^"1 and a seeding density of 8000 cells- cm^"2 was suitable. The Plastic Plus microcarriers were suitable for the expansion of the hUC MSCs, but not for bead-to-bead transfer.

A potential problem of microcarrier-based expansion is the cell number determination. For this reason an online monitoring system like dielectric spectroscopy should be chosen for the expansion of hUC MSCs in a large scale stirred tank bioreactor. A medium optimization towards to a high glucose medium could be carried out to prevent a medium exchange. Another possibility is to establish a controlled and monitored glucose feed, with regard that the cell growth get not affected by toxic levels of lactate and ammonium.

In the static and dynamic culture of hUC MSCs, there was no difference in cell growth and metabolism between cells from male and female donors. On one hand, this indicates a successful process transfer from static to dynamic system, and on the other hand that the age and source of the cells has more influence than the gender. In fact, that MSCs derived from umbilical cord are very young with a high proliferation activity and the advantages of cell isolation make these cells a good alternative to MSCs derived from bone marrow.

Claims

A method of producing isolated umbilical cord mesenchymal stem cells

(a) incubating umbilical cord tissue with a digestive solution to yield digested umbilical cord tissue;

(b) dissociating said digested umbilical cord tissue to yield a dissociated umbilical cord tissue solution comprising umbilical cord mesenchymal stem cells dispersed in tissue debris;

(c) separating said umbilical cord mesenchymal stem cells from said dissociated umbilical cord tissue solution to yield separated umbilical cord mesenchymal stem cells;

(d) suspending said separated umbilical cord mesenchymal stem cells in a suspension comprising a quantity of xenogen-free culture medium and red blood cell lysing solution, said xenogen-free culture medium comprising human platelet lysate; and

(e) isolating said umbilical cord mesenchymal stem cells from said culture medium and said red blood cell lysing solution, to yield said isolated umbilical cord mesenchymal stem cells.

2. The method of claim 1, wherein said quantity of xenogen-free culture medium comprises from about 2% to about 20% by volume of human platelet lysate.

3. The method of claim 1, wherein blood vessels are not removed from said umbilical cord tissue prior to said incubating (a).

4. The method of claim 1, wherein said incubating (a) occurs at a temperature of from about 30°C to about 45°C.

5. The method of claim 1, wherein said incubating (a) occurs for less than 10 hours.

6. The method of claim 1, wherein said umbilical cord tissue is treated with an antiseptic solution prior to said incubating (a).

7. The method of claim 1, wherein said separating (c) comprises filtering said dissociated umbilical cord tissue solution to remove said tissue debris, and pelleting said umbilical cord mesenchymal stem cells by centrifugation.

8. The method of claim 1, wherein said incubating (a) and said dissociating (b) are carried out in the same container.

9. The method of claim 1, wherein said xenogen-free culture medium is essentially free of fetal bovine serum.

10. The method of claim 1, further comprising expanding said isolated umbilical cord mesenchymal stem cells by:

(f) adding said isolated umbilical cord mesenchymal stem cells to a tissue culture plate or seeding said isolated umbilical cord mesenchymal stem cells on a microcarrier, said tissue culture plate or said microcarrier comprising a second quantity of xenogen-free culture medium comprising human platelet lysate;

(g) incubating said isolated umbilical cord mesenchymal stem cells in said tissue culture plate or on said microcarrier to yield cultured umbilical cord mesenchymal stem cells; and

(h) transferring said cultured umbilical cord mesenchymal stem cells to a second tissue culture plate or to a second microcarrier, to yield expanded umbilical cord mesenchymal stem cells.

11. The method of claim 10, wherein said second quantity of xenogen-free culture medium comprises from about 2% to about 20% by volume of human platelet lysate.

12. The method of claim 10, wherein said transferring (h) occurs after said cultured umbilical cord mesenchymal stem cells achieve at least about 70% confluency.

13. The method of claim 10, wherein said isolated umbilical cord mesenchymal stem cells are added to said tissue culture plate at a level of about 5,000 to about 20,000 live cells per cm².

14. The method of claim 10, wherein said microcarrier comprises a modified polystyrene or cross-linked polystyrene surface.

15. The method of claim 14, wherein said surface comprises a cationic charge, or is coated with high silica glass or recombinant RGD-containing protein.

16. The method of claim 15, wherein the surface is cross-linked polystyrene with a cationic surface charge.

17. The method of claim 1, further comprising cryopreserving said isolated umbilical cord mesenchymal stem cells.

18. The method of claim 17, wherein said isolated umbilical cord mesenchymal stem cells are crysopreserved in a cryopreservative medium with human platelet lysate.

19. The method of claim 1, wherein said digestive solution is a digestive enzyme solution.

20. A xenogen-free culture medium comprising glucose, glutamine, human platelet lysate, and heparin.

21. The culture medium of claim 20, further comprising an antibiotic-antimycotic component.

22. The culture medium of claim 20 consisting essentially of said glucose, said glutamine, said human platelet lysate, and said heparin.