WO2009052209A2

WO2009052209A2 - Isolation of stem cells and effective control of contamination

Info

Publication number: WO2009052209A2
Application number: PCT/US2008/080039
Authority: WO
Inventors: Kathy E. Mitchell; Steven M. Hoynowski
Original assignee: University Of Kansas
Priority date: 2007-10-16
Filing date: 2008-10-15
Publication date: 2009-04-23
Also published as: WO2009052209A3

Abstract

The invention relates to the improved isolation and culture of stem cells. More particularly the invention relates to a method that provides for increased recovery of sterile and viable stem cells from umbilical cord and other tissue sources where stem cells are not readily dissociated.

Description

ISOLATION OF STEM CELLS AND EFFECTIVE CONTROL OF

CONTAMINATION

BACKGROUND OF THE INVENTION

Field of the Invention The invention relates to the improved isolation and culture of stem cells from amniote species (potentially any animal with an umbilical cord, including humans). More particularly the invention relates to a method that provides for a greatly improved recovery of sterile and viable stem cells from umbilical cord or other biological sample where stem cells are not readily dissociated, such as liver, extraembryonic tissues, intestinal lining, or body fluids which may require special handling to obtain sterile, viable cells, including synovial and amniotic fluid. The stem cells isolated may be at least multipotent and may be totipotent or nearly totipotent and are envisaged to have a variety of end uses. The cells are derived from a readily available source that is not controversial in humans or other animal applications.

Description of the Related Art

The proposal that stem cells be obtained from an embryo source (commonly fertilized egg cells from fertility clinics) remains ethically controversial. The controversy surrounding obtaining stem cells from newly fertilized human material has increased a need for obtaining useful stem cells from a non-controversial source. Accordingly, a substantial need for obtaining stem cells having a powerful, universal and versatile treatment capability is present.

Stem cells can be isolated from any known source of stem cells, including, but not limited to, bone marrow, both adult and fetal, mobilized peripheral blood (MPB) and umbilical cord blood. The use of umbilical cord blood is discussed, for instance, in lssaraghshi et al. (1995) N. Engl. J. Med. 332:367-369. Initially, bone marrow cells can be obtained from a source of bone marrow, including but not limited to, ileum (e.g. from the hip bone via the iliac crest), tibia, femora, spine, or other bone cavities. Other sources of stem cells include, but are not limited to, embryonic yolk sac, fetal liver, and fetal spleen. Other mature tissue sources have been proposed as sources of stem cells, however these tissues are as yet not demonstrated to be workable.

Human pluhpotent cells have been developed from two sources with methods previously developed in work with animal models. Pluhpotent stem cells have been isolated directly from the inner cell mass of human embryos (ES cells) at the blastocyst stage obtained from In Vitro Fertilization programs. Pluripotent stem cells (EG cells) have also been isolated from terminated pregnancies.

Multipotent stem cells have been found in adult tissue. For example, blood stem cells, found in the bone marrow and blood stream of adults, continually replenish red blood cells, white blood cells and platelets. However, as a source for therapeutically useful or pluripotent stem cells, adults tissue sources remain problematic. Stem cells have not been isolated from all body tissues. Even when present in a tissue, adult stem cells are often present in only minute numbers and are difficult to isolate and purify. There is evidence that such adult stem cells may not have the same capacity to adapt or proliferate or differentiate as younger cells obtained from blastocyst, fetal or neonatal sources. Research on the early stages of cell specialization may not be possible with more mature and specialized adult stem cells. In addition, obtaining stem cells from most adult tissues requires an invasive procedure which is in itself problematic and also increases the risk of problems with sterility.

Pluripotent Stem Cells-Applications : i. Research:

Pluripotent stem cells have a number of possible applications. Pluripotent stem cells could provide insight into the complex events of human development particularly the cellular decision-making process that results in cell specialization. This might suggest treatments for disorders of abnormal cell specialization such as cancer and birth defects. Generating pluripotent stem cells would be useful for generating transgenic non-human primates for models of specific human genetic diseases or for other purposes. Stem cells will allow the generation of models for any human genetic disease for which the responsible gene has been cloned. The human genome project will identify an increasing number of genes related to human disease, but will not always provide insights into gene function. Transgenic models will be essential for elucidating mechanisms of disease and for testing new therapies. ii. Drug Testing:

Drug testing may benefit from a source of human pluripotent stem cells as new medications could be tested on human cell lines before animal and human research, iii. Cell Therapies: Many diseases are the result of disruption of cellular function or destruction of body tissues. Stem cells could be used in "cell therapies" to replace destroyed, non-functioning or abnormally functioning tissue. For example, recent studies have demonstrated that neural stem cells from the Central Nervous System (CNS) show tropism for specific diseased areas of the brain when grafted into animals. Neural stem cells from the CNS are rare, difficult to obtain and are not a feasible source of cells for applications in human medicine. In the mid-1990's, it was shown that embryonic stem cells from mice could be induced to form neurons and glia in vitro. If pluripotent stem cells can be stimulated to develop into specialized cells, they could be used to treat a range of Central Nervous System disorders such as Parkinson's and

Alzheimer's disease, spinal cord injury, stroke, ALS, Hematopoietic Disorders such as sickle cell disease, leukemia, Cardiac Disorders, inborn metabolic and storage diseases and other diseases, for example, diabetes.

By manipulating culture conditions, stem cells can be induced to differentiate to specific cell types such as blood cells, neural cells or muscle cells to mention a few examples. iv. Tissue Growth and Transplantation:

Transplantation of exogenous progenitor cells may provide a means to repopulate diseased tissues and organs. One source of exogenous progenitor cells has been Bone Marrow Stromal (BMS) cells. BMS cells are pluhpotent cells that can differentiate into bone, cartilage, fat, muscle, tendon, neurons and many other tissues. BMS cells transplanted into rats with induced liver damage contribute to the formation of new hepatic oval cells that can further differentiate into hepatocytes and ductal epithelium. Bone marrow derived cells also ^'home^' in to damaged muscle in irradiated mice. BMS cells injected intracerebroventricular migrate extensively and differentiate into glial cells and neurons in neonatal mice. Spinal cord neural stem cells injected into the Central Nervous System (CNS) differentiate into neurons or glia depending upon the injection site. Like the ^'homing^' potential of BMS cells to damage e.g. liver or muscle, neural stem cells and embryonic neuroblasts have tropism for glioma or degenerating neurons in adult brains. Neuroblasts injected into cortical lesions differentiate into projection neurons containing the appropriate neurotransmitter and receptor phenotype.

While the technique of ^'Tissue transplantation^' has been utilized extensively in order to replace damaged organs or tissues, problems with the procedure continue to limit its use. Finding donors is a problem. Harvesting the tissue (or cells) involves an invasive procedure. The supply of tissue is limited and patients often have to wait for long intervals before an organ is available. Some organs cannot be transplanted. The recipient must be immune- suppressed to a degree that can have undesirable side effects and furthermore makes the patient susceptible to infections. The use of fetal tissues has raised ethical concerns. Sophisticated banking or storing materials for transplant is necessary. Post-mitotic cells are not amenable to genetic manipulation. In many applications, a strong need for culture technology capable of growing and maintaining stable or useful cultures of stem cells has been a highly desired end. Many current stem cell cultures are based on murine cell culture "feeder cell" technology. Non-species specific feeder cell technology reduces the value of stem cell cultures due to the foreign nature of the source of the feeder cell. This is true for number of reasons including the fact that such non-species specific feeder cells contain both foreign cells and foreign growth factors. Further, it is thought that the use of non-species specific feeder cells in combination with different but desirable cultured cells cannot provide the optimum growth conditions as species specific derived feeder cells. This issue is particularly relevant to agricultural animals, endangered species, laboratory animals and non-human primate cells. Still further, non-human feeder cell technology reduces the value of human derived stem cell cultures. This is true for number of reasons including the fact that such non-human feeder cells contain both non-human cells and non-human growth factors. Further, we believe that the use of non-human feeder cells in combination with human cultured cells cannot provide the optimum growth conditions as human derived feeder cells. A method is necessary that would make stem cells, both pluhpotent and multipotent, easy to procure particularly in a manner that provides powerful, universal and versatile treatment capability using a commonly available non-controversial stem cell source. The process of isolating stem cells from the umbilical cord matrix and other tissues where stem cells are not readily dissociated has not been well established or particularly productive. One particular problem with umbilical cord derived cells and cells derived from other similar tissues, is microbial contamination and the inability to consistently and effectively establish sterile, viable cell cultures. Thus, there remains a need in the art to establish consistent methods for effective isolation and culture of these medically important sources of stem cells.

The present invention provides this and other advantages.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention provides a method for obtaining antimicrobial control in a cell culture comprising, isolating cells from a biological sample; plating the cells over a surface area of culture vessel such that antimicrobial control is obtained, wherein the surface area of culture vessel required for obtaining antimicrobial control is determined by the formula 8.2- 12.4 cm² surface area of culture vessel/g of biological sample; and wherein a volume of culture media needed for plating the cells over the determined surface area of culture vessel is determined using a culture vessel manufacturer's minimum recommended volume of media for the culture vessel. In one embodiment, the cells are umbilical cord matrix stem cells and the biological sample comprises umbilical cord. Other biological samples contemplated for use in the methods described herein include, but are not limited to liver, extraembryonic tissue, intestinal lining, adipose tissue, decidual teeth, brain tissue, synovial fluid or amniotic fluid.

In one embodiment of the methods described herein, the biological sample is first triturated through a mesh screen following a treatment with hyaluronidase and collagenase. In one embodiment, the microbial contamination is observed in 50% or fewer of culture vessel wells. In other embodiments, microbial contamination is observed in 40%, 30%, 20%, 10%, or fewer of culture vessel wells.

Another aspect of the invention provides a method for obtaining antimicrobial control in a culture of umbilical cord matrix cells comprising, a) triturating an umbilical cord tissue sample through a mesh screen; b) treating the triturated umbilical cord tissue sample with hyaluronidase and collagenase; c) isolating the umbilical cord matrix cells from the treated umbilical cord tissue sample; d) plating the cells over a surface area of culture vessel such that antimicrobial control is obtained, wherein the surface area of culture vessel required for obtaining antimicrobial control is determined by the formula 8.2- 12.4 cm² surface area of culture vessel/g of umbilical cord tissue sample; and wherein a volume of culture media needed for plating the cells over the determined surface area of culture vessel is determined using a culture vessel manufacturer's minimum recommended volume of media for the culture vessel. In one embodiment, microbial contamination is observed in 50% or fewer of culture vessel wells. In further embodiments, microbial contamination is observed in 40%, 30%, 20%, 10%, or fewer of culture vessel wells.

BRIEF DESCRIPTION OF THE DRAWING(S) Figure 1 is a graph showing a positive correlation between the percentage of uncontaminated wells with adherent cells and initial culture density (cm²/g of cord) (r = 0.80).

DETAILED DESCRIPTION OF THE INVENTION

The methods of isolation and culture of umbilical cord matrix cells, or stem cells from other tissues where stem cells are not readily dissociated, of the present invention greatly improves the recovery of sterile viable stem cells from umbilical cord and other tissue sources. Amelioration of bacterial contamination is achieved from the trituration or maceration process and culture of tissue by allowing the bacterial concentration to fall into the range of antibiotic control such that a population of bacteria or other microbe is too low to effectively increase in numbers. As described further herein, to achieve this goal, the processing fluid is increased to a volume to mass ratio of approximately 0.8 {e.g., the cells are plated using the formula 8.2-12.4 cm²/g of initial umbilical cord or other tissue or biological sample). This is an effective method to control bacteria or other microbes while at the same time allowing the formation of colonies of mesenchymal or other primitive stem cells from umbilical cord matrix or other sources listed above. This method greatly enhances the recovery of sterile, viable cells.

Using the methods described herein, stem cells may be isolated from the umbilical cord matrix of numerous different species or from other stem cell sources where the stem cells are not readily dissociated (e.g., liver, extraembryonic tissue, intestinal lining, brain, and the like or body fluids which may require special handling to obtain sterile, viable cells, including synovial and amniotic fluid) where microbial contamination may occur, with consistently improved recovery of sterile, viable cell cultures suitable for any of a variety of therapeutic applications. This method is particularly useful for generating cells for use in therapeutic settings as the occurrence of microbial contamination is reduced and the ability to wean the cultures from antibiotics in preparation for therapeutic application is increased.

As used herein, "obtaining antimicrobial control" refers to controlling the growth of microbes in a certain percentage of cell culture wells. In certain embodiments, obtaining antimicrobial control is achieved when fewer than 60% of the culture vessel wells have microbial contamination. In a further embodiment, obtaining antimicrobial control is achieved when fewer than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41 %, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of the culture vessel wells have microbial contamination. In one embodiment, obtaining antimicrobial control is achieved when the % of culture vessel wells with microbial contamination is 0%.

The cell cultures as described herein may be tested for microbial growth using any of a variety of assays and techniques known in the art. Illustrative assays include PCR detection of microbes, microbial growth/culture assays, detection of microbial proteins using ELISA or other similar detection techniques. Illustrative techniques are described, for example, in US Patents 4,563,419; 5,340,747; 5,366,867; 6,365,368; 6,468,743. Any of variety of pathogens may be tested for, including but not limited to Pseudomonas sp, Staphylococcus sp., Streptococcus sp., Bacillus sp., Escherichia coli,

Mycoplasma, and the like. Any of a variety of kits is commercially available for cell culture contamination detection and is contemplated for use herein.

The isolation and culture technique described herein is illustrated using umbilical cord as a source of stem cells. As would be recognized by the skilled artisan, the methods described herein may be applied to other tissues sources of stem cells where the stem cells may not be readily dissociated from the tissue, such as, but not limited to, liver, extraembryonic tissue, intestinal lining, brain and the like and stem cells from body fluids which may require special handling to obtain sterile, viable cells, including synovial and amniotic fluid.

Isolation and Culture of Umbilical Cord Matrix (UCM) cells.

Stem cells are capable of self-regeneration and can become lineage committed progenitors which are dedicated to differentiation and expansion into a specific lineage. Following fertilization of an egg by a sperm, a single cell is created that has the potential to form an entire differentiated multi-cellular organism including every differentiated cell type and tissue found in the body. This initial fertilized cell, with total potential is characterized as totipotent. Such totipotent cells have the capacity to differentiate into extra-embryonic membranes and tissues, embryonic tissues and organs. After several cycles (5 to 7 in most species) of cell division, these totipotent cells begin to specialize forming a hollow sphere of cells, the blastocyst. The inner cell mass of the blastocyst is composed of stem cells described as pluripotent because they can give rise to many types of cells that will constitute most of the tissues of an organism (not including some placental tissues etc.). Multipotent stem cells are more specialized giving rise to a succession of tissue-specific progenitor cells which then give rise to mature functional cells. The multipotent stem cell can give rise to mesodermal, ectodermal and endodermal progenitors. Thus, the hierarchy of stem cells is: totipotent stem cells → pluripotent stem cells → multipotent stem cell → committed cell lineage (progenitor and mature cell-types).

True pluripotent stem cells should: (i) be capable of indefinite proliferation in vitro in an undifferentiated state; (ii) maintain a normal karyotype through prolonged culture and cryogenic storage; and (iii) maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture. Strong evidence of these required properties have been published only for rodent embryonic stem cells (ES cells) and embryonic germ cells (EG cells) including mouse (Evans & Kaufman, Nature 292: 154-156, 1981 ; Martin, Proc Natl Acad Sci USA 78: 7634-7638, 1981 ) hamster (Doetschman et al. Dev Biol 127: 224-227, 1988), and rat (lannaccone et al. Dev Biol 163: 288-292, 1994), and less conclusively for rabbit ES cells (Giles et al. MoI Reprod Dev 36: 130-138, 1993; Graves & Moreadith, MoI Reprod Dev 36: 424-433, 1993). However, only established stem cell lines from the rat (lannaccone, et ai, 1994, supra) and the mouse (Bradley, et al., Nature 309: 255-256, 1984) have been reported to participate in normal development in chimeras.

The present invention provides methods for isolating and culturing umbilical cord matrix (UCM) stem cells that results in vastly decreased contamination of the cultures. UCM can be isolated using techniques known in the art, such as described in US Patent No. 5,919,702 and US Patent

Application Publication No. 20040136967. Umbilical Cord Matrix (UCM) stem cells are also known as Wharton's Jelly Cells. Such cells can be found in nearly any animal with an umbilical cord, including amniotes, placental animals, humans, and the like. Such matrix cells typically include extravascular cells, mucous-connective tissue {e.g., Wharton's Jelly) but typically do not include cord blood cells or related cells. Any of these cells may provide a source for differentiated cells and can provide an important feeder environment for the establishment or maintenance of stem cell cultures. UCM stem cells derived from umbilical cord tissue can be isolated, purified and culturally expanded. UCM cells are isolated from a non-blood tissue specimen from umbilical cord containing UCM cells. The UCM cells are then added to a medium which contains factors that stimulate UCM cell growth without differentiation and allows, when cultured, for the selective adherence of the UCM stem cells to a substrate surface. The specimen-medium mixture is cultured and the non-adherent matter is removed from the substrate surface. The use of umbilical cord blood is also discussed, for instance, in lssaragrishi et al. (1995) N. Engl. J. Med. 332:367-369.

The UCM stem cells of the invention are isolated from umbilical cord sources, preferably from Wharton's jelly. Wharton's jelly is a gelatinous substance found in the umbilical cord which has been generally regarded as a loose mucous connective tissue, and has been frequently described as consisting of fibroblasts, collagen fibers and an amorphous ground substance composed mainly of hyaluronic acid (Takechi et al., 1993, Placenta 14:235-45). Various studies have been carried out on the composition and organization of Wharton's jelly (Gill and Jarjoura, 1993, J. Rep. Med. 38:611-614; Meyer et al., 1983, Biochim. Biophys. Acta 755:376-387). One report described the isolation and in vitro culture of "fibroblast-like" cells from Wharton's jelly (McElreavey et al., 1991 , Biochem. Soc. Trans. 636th Meeting Dublin 19:29S).

Umbilical cord is generally obtained immediately upon termination of either a full term or pre-term pregnancy. For example, but not by way of limitation, the umbilical cord, or a section thereof, may be transported from the birth site to the laboratory in a sterile container such as a flask, beaker or culture dish, containing sterile isotonic buffered saline solution or a medium, such as, for example, Dulbecco's Modified Eagle's Medium (DMEM). The umbilical cord is preferably maintained and handled under sterile conditions prior to and during collection of the Wharton's jelly, and may additionally be surface-sterilized by brief surface treatment of the cord with, for example, a 70% ethanol solution, followed by a rinse with sterile, distilled water. The umbilical cord can be briefly stored for up to about three hours at about 3-5° C, but not frozen, prior to extraction of the Wharton's jelly. Wharton's Jelly is collected from the umbilical cord under sterile conditions. In one embodiment, the cord is cut transversely with a scalpel, for example, into approximately one inch sections, and each section is transferred to a sterile container containing a sufficient volume of phosphate buffered saline (PBS) containing CaCI₂ (0.1 g/l) and MgCI₂6H₂O (0.1 g/l) to allow surface blood to be removed from the section by gentle agitation. The section is then removed to a sterile-surface where the outer layer of the section is sliced open along the cord's longitudinal axis. The blood vessels of the umbilical cord (two veins and an artery) are dissected away, for example, with sterile forceps and dissecting scissors, and the umbilical cord is collected and placed in a sterile container, such as a 100 mm TC-treated Petri dish. The umbilical cord may then be cut into smaller sections, such as 2-3 mm³ for cultuhng. Another method relies on enzymatic dispersion of Wharton's Jelly with collagenase and/or hyaluronidase, or other appropriate enzyme known to the skilled artisan, and isolation of cells by centrifugation followed by plating.

Wharton's jelly is incubated in vitro in culture medium under appropriate conditions to permit the proliferation of any UCM cells present therein. Any appropriate type of culture medium can be used to isolate the UCM cells of the invention, such as, but not limited to, DMEM, McCoys 5A medium (Gibco), Eagle's basal medium, CMRL medium, Glasgow minimum essential medium, Ham's F-12 medium, Iscove's modified Dulbecco's medium, Liebovitz' L-15 medium, and RPMI 1640, among others. The culture medium may be supplemented with one or more components including, for example, fetal bovine serum (FBS), equine serum (ES), human serum (HS), defined serum substitutes such as ES-Knockout Serum Replacement® (Invitrogen, Carlsbad, CA) and one or more antibiotics and/or antimycotics to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination, among others.

Methods for the selection of the most appropriate culture medium, medium preparation, and cell culture techniques are well known in the art and are described in a variety of sources, including Doyle et al., (eds.), 1995, Cell and Tissue Culture: Laboratory Procedures, John Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991 , Animal Cell Bioreactors, Butterworth- Heinemann, Boston, which are incorporated herein by reference.

Culturing UCM cells involves fractionating the source of cells (Wharton's Jelly) into two fractions, one of which is enriched for stem cells and thereafter exposing the stem cells to conditions suitable for cell proliferation. The cell enriched isolate thus created comprises stem cells.

After culturing Wharton's Jelly for a sufficient period of time, for example, about 10-12 days, UCM derived stem cells present in the explanted tissue will tend to have grown out from the tissue, either as a result of migration therefrom or cell division or both. These UCM derived stem cells may then be removed to a separate culture vessel containing fresh medium of the same or a different type as that used initially, where the population of UCM derived stem cells can be mitotically expanded.

Alternatively, the different cell types present in Wharton's Jelly can be fractionated into subpopulations from which UCM derived stem cells can be isolated. This may be accomplished using standard techniques for cell separation including, but not limited to, enzymatic treatment to dissociate Wharton's Jelly into its component cells, followed by cloning and selection of specific cell types (for example, myofibroblasts, stem cells, etc.), using either morphological or biochemical markers, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal eluthation (counter-streaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis, fluorescence activated cell sorting (FACS) and magnetic bead based on selection for specific surface antigens. For a review of clonal selection and cell separation techniques, see Freshney, 1994, Culture of Animal Cells; A Manual of Basic Techniques, 3d Ed., Wiley- Liss, Inc., New York. In one particular embodiment, UCM cells are isolated and cultured as follows: umbilical cords are obtained from full term infants in accordance with the appropriate Human Subjects Approval. The human umbilical cord matrix (HUCM) cells are grown from umbilical cord tissue that was processed in the following manner: the cord is prepared for processing by rinsing in a 1000 ml_ beaker containing approximately 500 ml_ of 95% ethanol or sufficient amount to completely cover the cord, for 30 seconds. The cord is then flamed until the ethanol is dissipated, then washed thoroughly 2X, for 5 minutes, in cold sterile PBS (500 ml_). Next, the cord is submerged in 500 ml_ Betadine solution 1X for 5 minutes followed by rinsing thoroughly 2X for 5 minutes with cold sterile PBS (500 ml_) to remove the Betadine. The cord is then sectioned into ~5 cm pieces. When the cord piece has been completely dissected and cleaned of blood with PBS, it is placed into the 50 ml tube or 100 mm tissue culture plate containing 40U/ml_ hyaluronidase/0.4mg/mL collagenase solution for 30 minutes in a 37⁰C humidified incubator with 5% CO2. The digested piece of cord section is then placed into a sterilized cell strainer and pestle with a 40 mesh screen installed. As would be recognized by the skilled artisan, other sized mesh screens may also be appropriate and are contemplated for use herein. The apparatus is then placed on a sterile 100 mm Petri dish, and 5-10 ml_ of Defined Media (DM) is added which contains: 58% low glucose DMEM (Invitrogen, Carlsbad, CA), 40% MCDB201 (Sigma, St. Louis, MO), 1X insulin- transferhn-selenium-A (Invitrogen, Carlsbad, CA), 0.15 g/mL AlbuMAX I (Invitrogen, Carlsbad, CA), 1 nM dexamethasone (Sigma, St. Louis, MO), 100 μM ascorbic acid 2-phosphate (Sigma, St. Louis, MO), 100 U penicillin, 1000 U streptomycin (Mediatech, Inc., Herdon, VA), 2% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 10 ng/mL epidermal growth factor (EGF) (R & D Systems, Minneapolis, MN), and 10 ng/mL platelet-derived growth factor BB (PDGF-BB) (R & D Systems, Minneapolis, MN). The tissue is triturated and pushed through the strainer with a pestle until most of the tissue has lost its structure and the fluid is collected with a pipette. The sample is centrifuged at 750 RCF (x g) for 10 minutes. The media is aspirated off with care so as not to disturb the pellet. The pellet is resuspended in the appropriate volume of DM to obtain the desired range where antimicrobial control is obtained. In particular, in this embodiment, the cells are plated using the following formula: 8.2 - 12.4 cm²/g of original cord tissue sample. The cm² refers to the culture vessel surface area necessary for plating the cells isolated from the particular tissue sample (as measured in grams). The volume of media needed to plate the cells over that determined surface area is calculated based on recommended volumes of media for the particular cell culture vessel being used. For example: If the initial weight of cord tissue is 18.4 g:

Using the formula 12.4 cm²/g: 12.4 cm²/g x 18.4g = 228 cm²

If a 6-well plate is being used for cultuhng, each well is 9.5 cm², so: (228 cm²) / (9.5 cm²/well) = 24 wells

If you are using a 12-well plate, each well is 3.8 cm² so: (228 cm²) / (3.8 cm²/well) = 60 wells If you are using a 24-well plate, each well is 1.9 cm² so: (228 cm²) / (1.9 cm²/well) = 120 wells Generally, a minimum volume of media is used in each well according to manufacturer recommendations, such as 2 ml_ for 6-well, 1 ml_ for 12 well, 0.5 ml_ for 24 well, 0.2 ml_ for 96 well. Each plate manufacturer states recommended range of volumes for each vessel. For example, Corning recommends a range of 1.9 to 2.9 mL/well for the 6-well plates. After resuspending the cell pellet in the appropriate volume of media using the above formula applied to the type of plate being used, the diluted cell preparation was then seeded into 6-well plates or other vessels as appropriate.

The cells are placed in a 37⁰C humidified incubator with 5% CO2 and left undisturbed for -24 hours. 24-48 hours after isolation, non-adherent cells are removed by washing three times with sterile PBS. Fresh DM is changed every two days. When culture confluency of between 50-80% is reached the cells are harvested using 0.05% trypsin/0.53 mM EDTA solution and re-plated into a T25 culture flask for further expansion in DM. Cultures are maintained at the stated confluency (50-80%) for propagation. Cultures are maintained in a 37⁰C humidified incubator with 5% CO2. Cultures are replenished with fresh DM every 2-3 days.

In another embodiment for culturing UCM derived stem cells, Wharton's Jelly is cut into sections, such as section of approximately 1 -5 mm³, and placed in an appropriate dish, such as a TC-treated Petri dish containing glass slides on the bottom of the Petri dish. The tissue sections are then covered with another glass slide and cultured in a complete medium, such as, for example, Dulbecco's MEM plus 20% FBS; or RPMI 1640 containing 10% FBS, 5% ES and antimicrobial compounds, including penicillin G (100 ug/ml), streptomycin sulfate (100 ug/ml), amphotericin (250 ug/ml), and gentamicin (10 ug/ml), pH 7.4-7.6. The tissue is preferably incubated at 37-39° C and 5% CO₂ for 10-12 days. However, as would be recognized by the skilled artisan, the temperature, O₂ and CO₂ levels can be adjusted. For example the temperature may range from 32°-40°C and the CO2 level may range in certain embodiments from 2%-7%. The number of days in culture can also be adjusted from about 5, 6, 7, 8, or 9 to about 13, 14, 15, 20, 25 or more days. A further example of a defined media is DMEM, 40% MCDB201 , 1X insulin-transferrin-selenium (ITS), 1X linoleic acid-BSA, 10^~8 M dexamethasone, 10^~4 M ascorbic acid 2- phosphate, 100 U penicillin, 1000 U streptomycin, 2% FBS, 10 ng/mL EGF, 10 ng/mL PDGF-BB.

The medium is changed as necessary by carefully aspirating the medium from the dish, for example, with a pipette, and replenishing with fresh medium. Incubation is continued as above until a sufficient number or density of cells accumulates in the dish and on the surfaces of the slides. For example, the culture obtains approximately 70 percent confluence but not to the point of complete confluence. The original explanted tissue sections may be removed and the remaining cells are trypsinized using standard techniques. After trypsinization, the cells are collected, removed to fresh medium and incubated as above. The medium is changed at least once at 24 hr post-trypsin to remove any floating cells. The adherent cells remaining in culture are considered to be UCM-derived stem cells.

Once the stem cells have been isolated, the population is expanded mitotically. The stem cells should be transferred or "passaged" to fresh medium when they reach an appropriate density, such as 3X10⁴-cm² to 6.5X10⁴-cm², or, defined percentage of confluency on the surface of a culture dish. During incubation of the stem cells, cells can stick to the walls of the culture vessel where they can continue to proliferate and form a confluent monolayer. Alternatively, the liquid culture can be agitated, for example, on an orbital shaker, to prevent the cells from sticking to the vessel walls. The cells can also be grown on Teflon-coated culture bags.

In another embodiment, the desired mature cells or cell lines are produced using stem cells that have gone through a low number of passages, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 passages. However, in some embodiments, cells are maintained for more doublings, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or more than 100 population doublings. The invention contemplates that once stem cells have been established in culture, their ability to serve as progenitors for mature cells or cell lines can be maintained, for example, by regular passage to fresh medium as the cell culture reaches an appropriate density or percentage of confluency, or by treatment with an appropriate growth factors, or by modification of the culture medium or culture protocol, or by some combination of the above. According to the invention, UCM cells may be obtained from Wharton's jelly collected from a subject's own umbilical cord. Alternatively, it may be advantageous to obtain UCM stem cells from Wharton's jelly obtained from an umbilical cord associated with a developing fetus or newly-born child, where the subject in need of treatment is one of the parents of the fetus or child. Alternatively, because of the "extraembryonic" source of cells isolated from Wharton's jelly, immune rejection of the cells of the invention and/or any differentiated cells produced therefrom may be minimized. As a result, such cells may be useful as "ubiquitous donor cells" for the production of various types of differentiated cells for use in any subject in need thereof.

The isolation and culture methods described herein can also be applied to other cell isolation and culture settings, particularly settings where high rates of microbial contamination are observed. Thus, application of the methods described herein is contemplated for any cell culture setting where microbial contamination is observed. For example, the methods described herein may be applied to the isolation of stem cells from liver, extraembryonic tissue, intestinal lining, brain, and similar tissue samples or biological samples where stem cells may not be readily dissociated from the connective tissue or from body fluids which may require special handling to obtain sterile, viable cells, including synovial and amniotic fluid. Thus, umbilical cord matrix is an illustrative tissue/biological sample and application of the methods described herein are contemplated for use with other biological samples.

EXAMPLES

EXAMPLE 1

ISOLATION, EXPANSION AND CRYOGENIC PRESERVATION OF STERILE CULTURES OF UMBILICAL CORD MATRIX CELLS

This example describes the isolation and culture of sterile stem cell cultures from umbilical cord using a method that greatly improved the recovery of sterile viable cells. The Example further shows expansion and cryogenic preservation of these sterile cultures of umbilical cord.

1. Preparation of umbilical cord

The cord sample was weighed and measured. The weight recorded here is used below to calculate the appropriate amount of culture medium to achieve antimicrobial control. The cord was prepared by rinsing in a 1000 ml_ beaker containing approximately 500 ml_ of 95% ethanol or a sufficient amount to completely cover the cord for 30 seconds. The cord was then flamed until ethanol is dissipated. The sample was then washed thoroughly twice for 5 minutes, in cold sterile PBS (500 ml_). The cord was then washed in 500 ml_ Betadine solution once for 5 minutes followed by rinsing thoroughly twice for 5 minutes with cold sterile PBS (500 ml_) to remove the Betadine. The wash media was then sampled for future analysis of contamination. The cord was then sectioned into ~5 cm pieces. The piece was filleted down the middle to open the cord. The cord was further dissected in order to expose as much of the inner part as possible. The goal at this step is to maximize the surface area of the cord exposed to subsequent treatment by enzymatic digestion. The cord was rinsed with PBS as needed to remove excess blood. When the cord piece was completely dissected and cleaned of blood with PBS, it was placed into a 50 ml tube or 100 mm tissue culture plate containing 40U/ml_ hyaluronidase/0.4mg/ml_ collagenase solution for 30 minutes in a 37⁰C humidified incubator with 5% CO2. The digestion solution was sampled for future analysis of contamination.

2. Isolation of Cord Matrix Cells

Each cord piece was further dissected into ~1 cm pieces. The piece of cord section was then placed into a sterilized cell strainer and pestle with a 40 mesh screen installed. The apparatus was then placed on a sterile 100 mm Petri dish, and 5-10 ml_ of Defined Media (DM) was added. The cells were triturated and pushed through the strainer with the pestle until most of the tissue had lost its structure and the fluid was collected with a 10 ml_ pipette. The sample was centrifuge at 750 RCF (x g) for 10 minutes. The media was aspirated off being careful not to disturb pellet. The pellet was resuspended in the appropriate volume of media to obtain the desired range where antimicrobial control was obtained. The cells were plated using the following formula: 8.2-12.4 cm²/g of original cord tissue sample. The cm² refers to the culture vessel surface area necessary for plating the cells isolated from the particular tissue sample (as measured in grams). The volume of media needed to plate the cells over that determined surface area is calculated based on recommended volumes of media for the particular cell culture vessel that my be used. For example: If the initial weight of cord tissue is 18.4 g:

Using the formula 12.4 cm²/g:

12.4 cm²/g x 18.4g = 228 cm² total surface area necessary for plating

If a 6-well plate is being used for cultuhng, each well is 9.5 cm², so:

(228 cm²) / (9.5 cm²/well) = 24 total wells needed for plating If you are using a 12-well plate, each well is 3.8 cm² so: (228 cm²) / (3.8 cm²/well) = 60 wells needed for plating If you are using a 24-well plate, each well is 1.9 cm² so: (228 cm²) / (1.9 cm²/well) = 120 wells needed for plating

Generally, a minimum volume of media is used in each well according to manufacturer recommendations, such as 2 ml_ for 6-well, 1 ml_ for 12 well, 0.5 ml_ for 24 well, or 0.2 ml_ for 96 well. Each plate manufacturer states recommended range of volumes for each vessel. For example, Corning recommends a range of 1.9 to 2.9 mL/well for the 6-well plates.

After resuspending the cell pellet in the appropriate volume of media using the above formula applied to the type of plate being used, the diluted cell preparation was then seeded into 6-well plates or other vessels as appropriate. The cells were placed in a 37⁰C humidified incubator with 5% CO₂ and left undisturbed for -24 hours.

3. Plastic adherent selection

24-48 hours after isolation, non-adherent cells were removed by washing three times with sterile PBS. Fresh DM was added and changed every two days. When culture confluency of between 50-80% was reached the cells are harvested using 0.05% trypsin/0.53 mM EDTA solution and re-plated into a T25 culture flask for further expansion in DM. Cultures were maintained at the stated confluency (50-80%) for propagation. Cultures were maintained in a 37⁰C humidified incubator with 5% CO2. Cultures were replenished with fresh DM every 2-3 days.

4. Antibiotic wean

After cells were expanded for 4-5 passages in antibiotic- containing media, sterile cultures were obtained by weaning from antibiotics by sequential reduction. Initially antibiotics were reduced by 50% until the antibiotics were sequentially removed. The sterile cultures were maintained and expanded in antibiotic-free DM. 5. Cell preservation

Sterile cultures of matrix cells were maintained at 50-80% confluency and expanded to obtain sufficient cells for direct use or cryogenically stored at a cell density of 0.5E06/ml_ in medium containing 10% DMSO/90%

FBS in liquid nitrogen for future use.

Defined Media formulation

58% low glucose DMEM (Invitrogen, Carlsbad, CA), 40% MCDB201 (Sigma, St. Louis, MO), 1X insulin-transferrin-selenium-A (Invitrogen, Carlsbad, CA), 0.15 g/mL AlbuMAX I (Invitrogen, Carlsbad, CA), 1 nM dexamethasone (Sigma, St. Louis, MO), 100 μM ascorbic acid 2-phosphate (Sigma, St. Louis, MO), 50 U/mL penicillin, 50 ug/mL streptomycin (Mediatech, Inc., Herdon, VA), 2% fetal bovine serum (FBS) (HyClone, Logan, UT), 10 ng/mL epidermal growth factor (rhEGF) (R & D Systems, Minneapolis, MN), and 10 ng/mL platelet-derived growth factor BB (rhPDGF-BB) (R & D Systems, Minneapolis, MN).

Using the above-described methods, stem cells were isolated from the umbilical cord matrix of numerous different species with consistently improved recovery of sterile, viable cell cultures suitable for any of a variety of medical and research applications. This is accomplished by initiating the cultures of the matrix or other stem cell-types at a density that minimizes microbial contaminants in the tissue cell isolates to levels which can be eliminated by antibiotics initially with eventual removal to obtain sterile stem cell cultures that can be propagated in antibiotic-free medium.

Figure 1 shows an example of one such experiment using multiple umbilical cords demonstrating the correlation between the percentage of wells with uncontaminated, viable cultures vs cm2/g of cells plated in the wells. The cords were obtained from equine foals that were born at full term and processed within 48 hours of birth. The cords were treated and cells collected according to the method described herein. The data shown in Figure 1 and the data for determination of contamination frequency was based on cords from 8 different animals which were processed to isolate cells and plated at the density range shown in Figure 1 in 6 well plates with the total number of wells plated from each cord dependent upon the weight of the cord sections that were cultured (ranging from 48 to 108 wells).

It was found that the range of culture conditions that maximizes recovery of viable stem cell cultures can be quantified as the % percent of wells with uncontaminated adherent cells that can be expanded per the total number of wells plated and can be calculated with the formula 8.4 to 16.2 cm²/g of umbilical cord tissue. The percentage of contaminated cells increases as the cell density increases in initial cultures demonstrating the necessity of diluting the initial tissue isolate to obtain minimal microbial contamination. For culture densities in the optimal range for obtaining uncontaminated viable cultures (8.4 to 16.2 cm²/g), the mean cells/cm² plated was 731 7- 59 (sem) with a resulting contamination level of 14 ⁺/- 12 % (sem). At higher densities of cells in the initial cell cultures < 3 cells/cm² with a mean of 5016 7- 732 cells/cm², the level of contamination was increased by ~3-fold to 45 7- 17 % (sem). These results show that there is an optimal range of plating density for initial culture of matrix stem cells to obtain sterile, viable cultures from 100% of the cords. Obtaining sterile, viable cultures is dependent upon both cell density and plating in multiple wells so to increase the likelihood of obtaining wells that contain both adherent cells and a level of microbial contamination that can be eliminated by antimicrobial levels that are relatively low. Low antimicrobial levels allows the cultures to subsequently be more readily weaned from and maintained as sterile in the absence of antimicrobials in the culture medium. Thus, this method is particularly useful for generating cells for use in therapeutic settings as the occurrence of microbial contamination is reduced and the ability to wean the cultures from antibiotics in preparation for therapeutic application is increased. All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims

Claims

1. A method for obtaining antimicrobial control in a cell culture comprising, isolating cells from a biological sample; plating the cells over a surface area of culture vessel such that antimicrobial control is obtained, wherein the surface area of culture vessel required for obtaining antimicrobial control is determined by the formula 8.2-12.4 cm² surface area of culture vessel/g of biological sample; and wherein a volume of culture media needed for plating the cells over the determined surface area of culture vessel is determined using a culture vessel manufacturer's minimum recommended volume of media for the culture vessel.

2. The method of claim 1 wherein the cells are umbilical cord matrix stem cells and the biological sample comprises umbilical cord.

3. The method of claim 1 wherein the biological sample is selected from the group consisting of liver, extraembryonic tissue, intestinal lining, adipose tissue, decidual teeth, brain tissue, synovial fluid and amniotic fluid.

4. The method of claim 2 or claim 3 wherein the biological sample is first triturated through a mesh screen following a treatment with hyaluronidase and collagenase.

5. The method of claim 1 wherein microbial contamination is observed in 50% or fewer of culture vessel wells.

6. The method of claim 1 wherein microbial contamination is observed in 40% or fewer of culture vessel wells.

7. The method of claim 1 wherein microbial contamination is observed in 30% or fewer of culture vessel wells.

8. A method for obtaining antimicrobial control in a culture of umbilical cord matrix cells comprising, a. triturating an umbilical cord tissue sample through a mesh screen; b. treating the triturated umbilical cord tissue sample with hyaluronidase and collagenase; c. isolating the umbilical cord matrix cells from the treated umbilical cord tissue sample; d. plating the cells over a surface area of culture vessel such that antimicrobial control is obtained, wherein the surface area of culture vessel required for obtaining antimicrobial control is determined by the formula 8.2-

12.4 cm² surface area of culture vessel/g of umbilical cord tissue sample; and wherein a volume of culture media needed for plating the cells over the determined surface area of culture vessel is determined using a culture vessel manufacturer's minimum recommended volume of media for the culture vessel.

9. The method of claim 8 wherein microbial contamination is observed in 50% or fewer of culture vessel wells.

10. The method of claim 8 wherein microbial contamination is observed in 40% or fewer of culture vessel wells.

11. The method of claim 8 wherein microbial contamination is observed in 30% or fewer of culture vessel wells.