WO2011109717A1 - Methods and processes for collecting, preserving, and culturing adult stem cells obtained from lochia and related compositions of matter - Google Patents

Abstract

A method is provided for collecting and isolating stem-like cells from lochia. The method includes cryopreserving, thawing, and culturing isolated cells. The isolated lochia cells may be combined with a pharmaceutically-acceptable excipient, a cosmeceutical agent, a therapeutic agent, or a media.

Description

METHODS AND PROCESSES FOR COLLECTING, PRESERVING, AND CULTURING ADULT STEM CELLS OBTAINED FROM LOCHIA AND RELATED

COMPOSITIONS OF MATTER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority and benefit of U.S. Provisional Patent Application Serial No. 61/310,588, entitled "Methods and Processes for Collecting, Preserving, and Culturing Adult Stem Cells Obtained from Lochia and Related Compositions of Matter" filed March 4, 2010, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] The present invention relates generally to collecting and preserving adult stem cells. In particular, the present invention relates to collecting lochia, isolating adult stem cells from the lochia, and preserving and culturing the adult stem cells and compositions of matter comprising the same.

Background of the Art

[0003] Lochia is post-partum vaginal discharge, containing blood, mucus, and placental tissue, including mesenchymal and epithelial cells. Lochia discharge typically continues for 4 to 6 weeks after childbirth and progresses through three stages. Lochia rubra is the first discharge, red in color because of the large amount of blood it contains. It typically lasts no longer than 3 to 5 days after birth. Lochia serosa is the term for lochia which has thinned and turned brownish or pink in color. It contains serous exudate, erythrocytes, leukocytes, and cervical mucus. This stage continues until around the tenth day after delivery. Lochia alba is whitish or yellowish-white. It typically lasts from the second through the third to sixth week after delivery. It contains fewer red blood cells and is mainly made up of leukocytes, epithelial cells, cholesterol, fat, and mucus.

[0004] Stem cells inherently possess the capability to undergo cellular division and cellular differentiation in vivo by way of control of cell-to-cell contact and intrinsic signals. Stem cells have been shown to be capable of dividing and differentiating in vitro into a variety of cells by controlling cell contact and intrinsic signals by stimulation with local environmental factors. It is recognized that stem cells may be obtained from several sources including a variety of adult tissues, such as bone marrow and also embryonic tissues.

[0005] Stem cells express the antigenic factor CDl 17 that is also known as a c-kit receptor, Steel factor receptor, and stem cell factor receptor. The gene for c-kit encodes a tyrosine kinase growth factor receptor for Stem Cell Factor (SCF), which is also known as mast cell growth factor and is essential for hematopoiesis, melanogenesis, and fertility. It is recognized that CDl 17 is expressed in hematopoietic stem cells, mast cells, germ cells, melanocytes, certain basal epithelial cells, luminal epithelium of the breast, and the interstitial cells of Cajal of the gastrointestinal tract. CDl 17 imparts a critical role in germ cell establishment, maintanence, and function. Research indicates that in the embryonic gonad, CDl 17 and its corresponding ligand SCF, are essential for the primordial germ cell survival and proliferation. Additionally, research indicates that CDl 17 and its corresponding ligand SCF are essential for the gamete production in response to gonadotropic hormones. In other words, CDl 17 in combination with the ligand SCF are necessary for the survival and proliferation of germ cells of the testis, the spermatogonia, and for the growth and maturation of oocytes. Research also indicates that CD117 is a potent growth factor for primitive hematopoietic cell proliferation in vitro.

[0006] Therapeutic applications for stem cells began with the early experimentation of bone marrow. Nearly a century ago, physicians administered bone marrow by mouth to patients with anemia and leukemia. Although such therapy was unsuccessful, laboratory experimenters eventually demonstrated that mice with defective marrow could be restored to health with infusions into the blood stream of marrow taken from other mice. This caused physicians to speculate whether it was feasible to transplant bone marrow from one human to another (allogeneic transplantation). Among the early attempts to do this were several transplants carried out in France following a radiation accident in the late 1950s for decades, stem cells from bone marrow and umbilical have been extensively used primarily in hematopoietic transplants to treat a wide variety of diseases. From the first stem cell transplants that utilized bone marrow, other sources for stem cells have been identified to include amniotic fluid, placenta, umbilical cord, cord lining, Wharton's Jelly, fat and in addition almost every organ in the human body has a small quantity of stem cells.

[0007] Effects of the human immune system, the body's inherent mechanism to defend itself from infection and foreign substances, became a critical consideration in early transplants as researchers increasingly encountered severe illness and/or fatalities resulting from the body's rejection of cells later characterized as Graft versus Host Disease (GVHD). Performing marrow transplants in humans was not attempted on a larger scale until after a French medical researcher made a critical discovery about the human immune system. In 1958 Jean Dausset described the first of many human histocompatibility antigens. These proteins, found on the surface of most cells in the body, are called human leukocyte antigens, or HLA antigens. The body's immune system uses these HLA antigens to identify which cells belong in the body and which do not. Whenever the immune system does not recognize the antigens on a cell, it creates antibodies and other substances to destroy the cell. Objects that the body looks for and destroys are infection-causing bacteria, viruses, tumor cells and foreign objects such as splinters. In this way, the immune system defends the body against things that can enter the body and cause harm. Depending upon the disease state targeted for treatment, autologous transplantation utilizing an individuals own stem cells may have certain potential advantages resulting from the benefits of an exact HLA match. The closer the donor's and recipient's HLA antigens match, the less likely it is that the T cells (immune system cells) of the donated marrow will react against the patient's body. After the discovery of the HLA system, medical researchers next turned their attention to determining what degree of HLA match was required to ensure successful transplants.

[0008] The transplantation of adult stem cells derived from bone marrow has been successfully used in treatment of human disease such as Fanconi's Anemia, Aplastic Anemia, Acute and Chronic Leukemias, Myeloproliferative Disorders, Myelodysplasia Syndromes, Lymphoproliferative Disorders and other malignancies. Alternative sources of bone marrow adult stem cells include peripheral blood progenitor cells, umbilical cord blood and mesenchymal stem cells harvested from these sources. However, there are several shortcomings associated with therapeutic use of adult stem cells. Adult stem cells have been shown to have limited efficacy such as slow growth and loss of pluripotency after several passages in culture.

[0009] Embryonic stem cells have demonstrated proliferative potential making them suitable for cellular therapy. However, human embryonic stem cells have been shown to produce teratomas. Additionally, the harvesting of stem cells from embryos poses ethical concerns due in part to the destruction of the growing embryo in the harvesting process. Other sources of stem cells have been identified that overcome at least some of the issues associated with adult and embryonic stem cells.

[0010] One source is umbilical cord blood that contains human adult stem cells. Umbilical cord blood has proven to be a viable source of stem cells for several reasons. Umbilical cord blood is relatively easy to procure during delivery of a child and to process for cryopreservation. Umbilical cord blood provides a suitable source of stem cells capable of cellular division and differentiation into a variety of cell types. Stem cells derived from cord blood have been used in clinical settings to treat several known human disorders and diseases as an alternative to bone marrow. In particular, stem cells derived from cord blood have been successful in hematopoietic engraftment and hematopoietic reconstitution. A case study demonstrates the use of umbilical cord blood in treating spinal cord injuries. Additionally, stem cells derived from cord blood have been shown in animal models to reverse the effects of stroke and myocardial infarction. Further research is being conducted to identify additional therapeutic uses of stem cells derived from cord blood.

[0011] Lochia cells have demonstrated a proliferative capacity in culture similar to embryonic stem cells. These cells also display associated mesenchymal stem cell markers known in the field and for these reasons it is believed that these cells will be able to also demonstrate pluripotent capability in vitro and in vivo. The use of lochia cells as a source of stem cells is beneficial for several reasons. The lochia cells are considered waste. Additionally, the process of procuring lochia cells after childbirth imparts little risk to the donor associated with the collection techniques. Furthermore, any ethical concerns or considerations are alleviated by collecting cells that are otherwise considered waste.

[0012] There are a variety of target cells which are purported to be obtained through the a variety of methodologies; however, there is none directed at this cell source nor do the references teach or suggest procurement of lochia cells and related processing and cell collection methodologies useful for obtaining a viable, maximum yield of lochia cells expressing the antigenic factor CD117 among other factors expressed.

[0013] Accordingly, there is a present need for methods to collect a suitable amount of lochia cells for ready shipment to a centralized laboratory for processing to isolate and to store the lochia cells. There is also a present need for methods to process lochia fluid, blood and tissue to obtain a heterogeneous cell preparation of lochia cells derived from the lochia blood, fluid, and tissue in accordance with the present study. There is also a further need to select, culture, isolate and cryopreserve lochia cells from the heterogeneous population of cells, such as for example, cells that may express any one or more of the following cell surface markers or intracellular markers including CD29, CD44, CD49e, CD54, CD73, CD90, CD105, CD1 17, CD133, CD166, HLA class I, SSEA-3, SSEA-4, Oct-4, nanog while not expressing any one or more of the cell surface markers CD 14, CD31, CD34, CD45 and HLA class II. There is yet a further need to cryopreserve the heterogeneous population of cells or the isolated cells obtained from the lochia blood, fluid, and tissue collection.

SUMMARY OF THE INVENTION

[0014] The present invention comprises methods and processes for collecting lochia, isolating adult stem cells from the lochia, and preserving and culturing the adult stem cells and compositions of matter comprising the same.

[0015] The present invention comprises methods to collect a suitable amount of lochia cells for ready shipment to a centralized laboratory for processing to isolate and to store the lochia cells.

[0016] The present invention comprises methods to process lochia fluid, blood and tissue to obtain a heterogeneous cell preparation of lochia cells derived from the lochia blood, fluid, and tissue in accordance with the present invention.

[0017] The present invention comprises methods to select, culture, isolate and cryopreserve lochia cells from the heterogeneous population of cells, such as for example, cells that may express most of the following cell surface markers or intracellular markers including CD29, CD44, CD49e, CD54, CD73, CD90, CD105, CD1 17, CD133, CD166, HLA class I, SSEA-3, SSEA-4, Oct-4, nanog and not expressing one or more of the cell surface markers CD 14, CD31 , CD34, CD45 and HLA class II.

[0018] The present invention comprises methods to cryopreserve the heterogeneous population of cells or the isolated cells obtained from the lochia blood, fluid, and tissue collection.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION [0019] Methods and processes are provided for collecting lochia, isolating adult stem cells from the lochia, and preserving and culturing the adult stem cells and compositions of matter comprising the same.

[0020] The phrase "lochia cell" is used generally to refer to stem-like cells that may express any combination of the following cell surface markers or intracellular markers including CD29, CD44, CD49e, CD54, CD73, CD90, CD105, CD117, CD133, CD166, HLA class I, SSEA-3, SSEA-4, Oct-4, nanog while not expressing one or more of the cell surface markers CD 14, CD31, CD34, CD45 and HLA class II and selected by the methods, processes and systems of the present invention.

[0021] The methods, processes and systems of the present study involve procurement of cells, isolation of cells, and separation, collection and cryopreservation of the cells.

[0022] Lochia Procurement

[0023] A collection kit may be used to collect lochia. The collection kit may comprise suitable devices for collecting a body fluid in appropriate collection vessels and shipping the collection vessels with the lochia, optionally in a media, to a processing facility for processing and storage.

[0024] A collection kit, which may be stored at room temperature, may comprise the following: a first collection tube pre-filled with a diluent to ensure the optimal conditions for transportation of the specimen, a second collection tube pre-filled with diluent; two foam brick cooling packs (one large and one small), which may be put into the freezer upon receipt of the kit; a lochia collection device with instructions on its use (the collection device may include a sanitary cupped pad or a sterile cup for catching larger volumes of discharge; eight antiseptic towelettes for use in preparing to collect the two samples; two ziplock style plastic bags one for each lochia sample collected in each of the first collection tube and the second collection tube; and a prepaid Federal Express (or courier paperwork) airbill for the return shipment of collected samples to a processing facility.

[0025] In an embodiment, a collection kit comprises a lochia transport container such as a box, bag or other container suitable for shipping lochia cells preferable at a cold temperature (for example Nanocool, Styrofoam or insulated container); a collection device (the device may comprise a sanitary pad having a cup inserted therein, a menstrual cup, a sterile urine cup, or a sterile cup of any kind that may be used to collect and transfer the lochia to the sterile collection tube containing diluent); a sterile container of suitable size including, but not limited to, a 50 ml container; Dulbecco's Phosphate Buffer Saline (DPBS) (Mediatech or equivalent including other media) that contains no calcium, magnesium or phenol red; the specimen may be collected with or without antibiotics which may include Cefazolin (0.5 micrograms per milliliter) and Streptomyocin (100 micrograms per milliliter) and/or Amphotericin B (2.5 micrograms per milliliter) or other suitable antibiotics; Heparin preservative-free which may be present at 10 units per milliliter of the media (or up 20 units per milliliter of media) or suitable anticoagulant (examples may include: ACD, CPD, CPDA, CPDA-1); plastic zipped bags with absorbent towels including a biohazard bag for the collected samples; sterile 4 x 4 gauze; sterile gloves; parafilm for wrapping the tube; cleaning wipe used prior to sample collection; completed according to instructions in the kit.

[0026] According to the invention, collection of the lochia may take into account steps to control excess contamination from microorganisms found normally on the skin and elsewhere on the body. The lochia may be collected during the heaviest flow of lochia for the highest cell yield. If possible, a donor of lochia should take a shower and wash thoroughly using soap before preparing to take a sample. Preparation for collection of lochia may also comprise removing any sanitary protection pads. The bladder may be emptied prior to collecting the lochia by the collection device. Hands may be washed thoroughly before and after collection of the lochia. The outer vaginal area may be cleaned using soap and water and then rinsed with water and allowed to dry. The outer vaginal area, perineum, and rectal areas may be cleansed thoroughly with one of the antiseptic towelettes included in the Collection Kit by wiping from the front of labia toward the rectum and discarding the wipe. This step may be repeated with a new wipe from the kit. The lochia collection device may be removed from its package for use. The lochia collection device may be placed directly at the vaginal opening without inserting the collection device into the vagina. The device should be left in place until an adequate sample of lochia is collected, which may be at least 1 ml. After an adequate sample of lochia is collected, the sample should be placed in the first collection tube containing diluent.

[0027] In an embodiment, the collection device may be placed according to instructions. After a period of time optimally less than 3 hours the collection device may be removed and the sample transferred to the sterile antibiotic-containing media.

[0028] All samples collected and placed in media should be stored between about 1 °C and 24°C and optimally between about 1°C to about 10°C post collection. When the sample is ready to ship the shipping container may be activated to cool the sample when using the Nanocool container or ice may be added to an insulted container. The samples should be kept cool in the container shipped or a refrigerator and should reach the lab within 72 hours of collection and preferable 48 hours.

[0029] The donor's hand may be washed using soap and water. The vaginal area may be cleansed with an antiseptic towelette in the collection kit. The sample of lochia in the collection device may be transferred to the first collection tube. Attention may be paid to avoiding contact between the collection device and the first collection tube. The first collection tube may be sealed. The first collection tube may be inverted several times to mix its contents. The first collection tube may be placed in one of the ziplock bags which is then sealed. The sealed bag with the first collection tube may be placed in a foam well of the collection kit. The first collection tube and foam brick may be placed in a refrigerator until an adequate sample may be collected in the second collection tube.

[0030] The collection steps may be repeated to collect any one or more samples of lochia which is placed in a second collection tube.

[0031] Shipment of Lochia

[0032] The first collection tube and the second collection tube, if a second lochia sample was collected, may be removed from the refrigerator and placed in the bottom of a large well in the foam lining of a box provided with the collection kit. The box containing the first collection tube and the second collection tube may be shipped using a suitable carrier for biological material.

[0033] The lochia transport container is closed, secured and sealed. Other suitable containers may be used for transportation of the sterilized container as long as the container maintains the lochia cell sample at a temperature between about 1 °C to about 15°C and preferably between about 1 °C to about 10°C during shipment. For example, other suitable containers include but are not limited to cooled and insulated shipping containers sold by Therapak Corporation, insulated containers of any type or a Styrofoam box designed for shipment. A carrier must be contacted within at least two (2) hours of shipment pick up of the lochia cell transport container. The carrier may be AirNet or other preferred courier suitable for transporting biological materials or other shipment companies such as FedEx or UPS. The lochia cell shipment container should arrive at the processing facility within about 24 hours to about 72 hours of collection. It is preferred that the lochia cell shipment container arrive at the processing facility between about 24 hours to about 48 hours after collection and particularly within about 48 hours after collection.

[0034] Processing Lochia Sample

[0035] Once received at the processing facility, the sterilized container is removed from the lochia cell shipment container, placed on ice in an ice pan (or the refrigerator), and transferred into a cleanroom in a biological safety cabinet (BSC) for lochia cell isolation, collection and preparation for cryopreservation.

[0036] Upon arrival at the processing facility, the lochia sample may be treated with antibiotics in an attempt to thwart microbiological contamination. The antibiotic treatment may occur through use of the following: HBSS, Vancomycin, 1 gm/vial, Claforan, 500 mg/vial, Amikacin, 1 gm/vial, Gentamycin, 1 gm/vial, Amphotericin B/Stabilizer - 250 μg/mL, Heparin-Preservative-Free (American Pharmaceutical Partners Inc.) concentration 1000 units per mL, Sterile 50 mL conical , 1 mL needle TB Syringe, 16-20 g luer lock needles, 12 cc syringe, 50 mL Tube rack, Red Biohazard bags in container, Red Biohazard sharps container, Regular trash bin, IPA - Isopropyl Alcohol used for disinfection typically at a 70 % concentration, Cavicide, Alcohol prep pads, Alcohol wipes, Sterile Gloves, 4 x 4 Gauze (sterile or autoclaved), and Vacuum pump with associated tubing, 500 mL/500 mL sterile filtration system. Equipment consists of Biological Safety Cabinet (BSC) and Centrifuge.

[0037] Preparation of Antibiotics

[0038] Antibiotics may be prepared with the following steps. Disinfect the septum of each antibiotic vial with sterile alcohol pads. Open new sterile alcohol pads and place on top of the septum of each antibiotic (touching the edges of the alcohol pad only). Prepare Antibiotic Media In a sterile container, add 3.2 mL of 100000 μg mL Vancomycin, add 4 mL of 250000 μg/mL of Claforan, add 4 mL of 100000 μg/mL of Amikacin, add 4.8 mL of 100000 μg/mL of Gentamycin, add 43.2 mL (10800 μg) of Amphotericin B. Total antibiotics will be added to 140.8 mL of HBSS to make a 20x concentrated antibiotic media, antibiotic media with 200 mL total volume. Vancomycin: Add 10 mL of HBSS to the 1 g vial to get a concentration of 100 mg/mL. Allow time for antibiotic to be reconstituted. Add 3.2 mL of 100 mg/mL concentration to the antibiotic cocktail to get a concentration of 1600 μg/mL. Claforan: With 2 vials, add 2 mL of HBSS to the 500 mg/vial to get a concentration of 250 mg/mL. Allow time for antibiotic to be reconstituted. Add 4 mL of the 250 mg/mL concentration to the antibiotic cocktail to get a concentration of 5000 μg/mL. Amikacin: Add 10 mL of HBSS to the 1 g vial to get a concentration of 100 mg/mL. Allow time for antibiotic to be reconstituted. Add 4 mL of 100 mg/vial concentration to the antibiotic cocktail to get a concentration of 2000 μg/mL. Gentamycin: Add 10 mL of HBSS to the 1 g vial to get a concentration of 100 mg/mL. Allow time for antibiotic to be reconstituted. Add 4.8 mL of 100 mg/mL concentration to the antibiotic cocktail to get a concentration of 2400 μg/mL. Amphotericin B: Add 43.2 mL of Amphotericin B (250 mg/mL) to the antibiotic cocktail to get a concentration of 54 μg/mL.

[0039] The concentrated media may be aliquoted out to 1 mL samples which will be later diluted to lx into the specimen. Add 0.4 mL of Heparin 1000 units/mL directly to the M2 specimen. Media must be stored at 2.0-8.0°C until used and has an expiration date of 56 days from the date of preparation.

[0040] Cell Processing

[0041] The disinfected lochia samples may be subjected to cell processing. General Processing Supplies may include (as used throughout whole process): Red biohazard bags and Red biohazard containers, Red biohazard sharps container, Regular trash bin, IPA - Isopropyl Alcohol used for disinfection typically at a 70% concentration, Cavicide or approved disinfectant, Alcohol prep pads, Alcohol wipes, Ice bin, Ice, Sterile Disposable Gloves, Non-sterile gloves, 4 x 4 Gauze (sterile or autoclaved), and Specimen Labels.

[0042] Processing Reagents and Materials may include: BacT/Alert blood culture bottle aerobic, BacT/Alert blood culture bottle anaerobic, 16-20 g luer lock needles, 12 cc Syringe, 10 mL pipettes, 50 mL tube rack, 50 mL tubes, 5 mL tube rack, Red top vacutainer tubes, 100 micron filter and Purple top K₂E EDTA K₂ Vacuette tube.

[0043] Cryopreservation Reagents and Materials may include: DPBS, DMSO, 25 % Human Serum Albumin, Cryoquat vials including top QC vial space (5 mL whose cap is made to hold 200 QC aliquots), 10 mL pipettes, 3 cc syringe, Testing Reagents and Materials, Non-sterile pipette tips, Non-sterile 5 mL tubes, Unopette, Trypan blue, Lens paper, Processing Equipment, Centrifuge, Centrifuge inserts, Biological Safety Cabinet

(BSC), Automated pipetor, Testing Equipment, Microscope, Hemacytometer, Pipetor -

20 μί, Cryopreservation Equipment, 1 mL pipette, Automated Pipetor, CRF - Cryomed

Controlled Rate Freezer or equivalent and Liquid Nitrogen LN₂ Storage Freezer.

[0044] Aseptic technique must always be used during processing.

[0045] All media and reagents are preferably placed on ice and are stored between about 2°C to about 8°C, but may also be about 1°C to about 15°C.

[0046] A buffered saline media (DPBS) is used throughout the lochia cell isolation process. The media comprises about 100 milliliters of DPBS, with about 2 ml of Heparin

(Heparin Sodium 1,000 USP Units/ml - American Pharmaceutical Partners).

[0047] Day 1 - Processing of specimen

[0048] Carefully wipe down the outside of the first collection tube prior to centrifugation. Ensure proper balance within the centrifuge prior to centrifugation. Centrifuge specimen at 2000 RPM for 7 minutes at 4.0°C. Carefully remove specimens from centrifuge, taking care not to disturb cell button. Carefully wipe down the container with Cavacide before placing under BSC.

[0049] Infectious Disease Testing

[0050] Carefully wipe down the outside of the second collection tube before placing it under BSC. Carefully wipe down the outside of two Purple top K₂E EDTA K₂ Vacuette tubes (or equivalent) and place it inside BSC. Fill both of the purple top tubes with 6 mL of the specimen from the second collection tube. If there are not enough specimens to fill both tubes with 6 mL, at least one needs to be completely filled with 6 mL. Remove the excess fluid above the pellet and record the volume of the excess fluid. Be careful not to get too close to the pellet as excessive cell loss will occur. Measure the volume of the pellet. The total sample volume equals the volume of the excess fluid plus the volume of the pellet. Re-suspend the pellet with HBSS to achieve a volume of about 8.6 mL. Pipette slowly up and down to determine if there are clots. If necessary, filter specimen utilizing a 100 micron filter into a new, sterile 50 mL conical tube. Make sure to put the proper identifying client labels on tube. Wash filter with 10 mL of HBSS in the tube containing the re-suspended pellet. If the sample does not need to be filtered, add the 10 mL of HBSS directly to the specimen. Add 1 mL of prepared antibiotic treatment and 0.4 mL of Heparin. Pipette slowly up and down to mix. Incubate at 2.0-8.0°C for 20-30 hours for antibiotic treatment. The samples may be placed on the rotator or inverted for the first 5 hours once an hour.

[0051] Day 2 - Processing of specimen

[0052] Centrifuge specimen at 2000 RPM for 7 minutes at 4.0°C. Remove the excess fluid above the pellet. Re-suspend the pellet with approximately 10 mL of DPBS, mix well (gently) and centrifuge. Centrifuge specimen at 2000 RPM for 7 minutes at 4.0°C.

[0053] In another embodiment, the lochia cells collected in a buffered saline conical collection tube are subjected to centrifugation to concentrate the lochia cells. Centrifugation comprises subjecting the lochia cells suspended in the buffered saline media to about 2000 rpm for about seven (7) minutes between about 4°C. The conical collection tubes are removed from the centrifuge and the supernatant is used for microbiological testing as described herein before the sample is manipulated. Any additional supernatant may be aspirated with suction and discarded. The pelleted cells may be resuspended at approximately 100 microliters of sample and removed for a cell count and viability. The cells may be incubated in antibiotics or if they were previously in antibiotic media they must be washed first and spun down again. The cells may then be prepared for cryopreservation or to take to a further processing step to concentrate the cells of interest.

[0054] In the BSC using sterile technique remove the top of the container to the lochia specimen. Obtain an aerobic blood culture bottle and an anaerobic blood culture bottle. Visually inspect each blood culture bottle prior to inoculation. Bottles with turbidity, signs of gas production and/or evidence of growth (i.e. yellow sensor) should not be used. Remove plastic flip tops from the aerobic and anaerobic culture bottles. Do not touch the septum. Disinfect the septum with sterile alcohol pads. Open new sterile alcohol pads and place on top of the septum (touching the edges of the alcohol pad only). Confirm the label on the blood culture bottles matches the labels on the specimen containers. Withdraw 5 mL of the specimen using a syringe. Inoculate 4 mL into the anaerobic blood culture bottle and 1 mL into the aerobic blood culture bottle.

[0055] Post Processing Testing

[0056] Remove the excess fluid above the pellet. Re-suspend the remainder of the cell suspension with DPBS to a final volume of 5.5 mL. Mix well. Pipette 500 μL of the well-mixed cell suspension to the bottom of a red top tube. Perform cell count and viability for the Manual Total Nucleated Cell Counts or automated by flow cytometry. Viability will be assessed using trypan blue or 7AAD. Place the remaining 5 mL specimen to chill on ice racks or ice for at least 15 minutes until ready to cyropreserve.

[0057] Cryopreservation [0058] The final cell product may be cryopreserved as follows: Add 3 mL DPBS to a client labeled 50 mL conical. Using a 3 cc syringe, add 1 mL of Human Serum Albumin into the 3 mL of DPBS. Chill for ten minutes on ice. Add 1 mL of 99 % DMSO to the Human Serum Albumin and DPBS. Chill the client specimen and cryopreservation media on ice for 15 minutes prior to addition of the cryopreservation. The DMSO Media may also be produced in a batch for the day as demonstrated in the table below. Final solution is 20 % HSA and 20 % DMSO in DPBS.

[0059] Place cryopreserved specimens in the transfer tank and transport the samples to the bunker for quarantine storage. Store all products in the vapor of Liquid Nitrogen at -

150.0°C or less.

[0060] In another embodiment, about five ml of heterogeneous cellular suspension obtained by centrifugation is combined with a cryopreservation agent in preparation for cryopreservation. The cryopreservation agent comprises a buffer, a protein, and a preservative. For example, the cryopreservation agent is about a five ml solution comprising about three ml of the buffered saline (DPBS), about one (1) ml of the protein HSA (Telacris Bio or other suitable protein), and about one (1) ml of the preservative DMSO (99% Stemsol). The cryopreservation agent may be made by first combining the desired volume of DPBS and HSA and chilling the mixture for about ten (10) minutes on ice, and then adding about one (1) ml of DMSO and chilling for about ten (10) minutes on ice. The cryopreservation agent is carefully added to the about five (5) ml of heterogeneous cellular suspension to a total volume of about ten (10) ml of mixture of heterogeneous cellular suspension and cryopreservation agent. The mixture may be separated into desired aliquot volumes in several vials adapted for cryopreservation. For example, the mixture is separated into five ml barcoded cryovials and some separate one ml QC vials adapted for cryopreservation or placed in cryovials containing a space in the cap for the QC sample called a Cryoquat(Nalgene).

[0061] Cryopreservation, Thawing, and Culturing

[0062] The mixture of a heterogeneous population of lochia cells and other cells and cryopreservation agent in the cryopreservation vials is subjected to several temperature reduction steps to reduce the temperature of the heterogeneous population of cells comprising lochia cells, and/or other cells to a final temperature of about -90°C utilizing a controlled rate freezer or other suitable freezer system (dump-freeze monitored or a freeze container (Nalgene)). The control rate freezers used will be Cryomed Thermo Forma Controlled Rate Freezer 7454 (Thermo Electron, Corp.). The following temperature reduction steps are programmed in the controlled rate freezer first reducing the mixture of the heterogeneous population of cells and cryopreservation agent to about 4°C and then reducing the mixture at about 1 °C per minute to about -3°C, and then about 10°C per minute to about -20°C, and then about 1°C per minute to about -40°C, and finally about 10°C per minute to about -90°C. Alternatively a program may be used that reduces the temperature of the mixture by approximately 1 to 2°C per minute. The cryovials containing the mixture of the heterogeneous population of cells and cryopreservation agent are placed in the controlled rate freezer or container and subjected to the temperature reduction steps or the container is subjected to a -80°C freezer. Once the mixture and cryopreservation agent reaches about -90°C, the cryopreservation vials are transferred to a cryogenic storage unit and stored in the vapor of liquid Nitrogen at a temperature at or below about -135°C or alternatively vials may be stored in the liquid phase of liquid nitrogen. For example, a suitable cryogenic storage unit includes, but is not limited to, LN2 Freezer MVE 1830 (Chart Industries).

[0063] Data relating to collection, processing, and storage of stem cells obtained from lochia according to the present invention is attached hereto as Table A.

[0064] Table A

[0065] In an embodiment, the cryopreserved cells may be thawed for any one or more of processing, culture, cell selection, and other use. After cells are removed from liquid nitrogen vapor, agitate the cryovial in 37°C water bath. Do not let cells completely thaw, it should include large ice pieces. Clean the vial completely with 70% alcohol or Cavacide. Transfer vial of partially thawed cells to chilled complete Chang's media with DNase. Mix gently by inversion. For a 5ml cell preparation use 25 ml of chilled media. When thawing cell lines it is recommended to add DNase; use 10 drops of DNase per 100ml of media. Spin in centrifuge at 120g for 5 minutes. Pipette off supernatant and dilute cells in Chang's complete media and wash one more time to remove residual DNase. Previously cultured cells should be plated at 2000/cm² with a 48 hour passage time but if more cells are plated they may be passaged in 24 hours. Use a T75 flask non- tissue culture treated to plate 150,000 in 15 ml of complete media. Cells may be incubated in a 5% C02 incubator until they are subconfluent to 70-80%. When cells are ready to be dissociated from the flask aspirate the media in the flask. The flask is then rinsed with 5 ml of phosphate buffered solution without calcium or magnesium. The PBS is removed after the attached cells have been washed once. About two ml of Trypsin-like enzyme (TrypLE) is added to a T75 flask (one ml for a T25 flask), pre-warmed at about 37°C and the flask is agitated to coat the cells with the enzyme. The flask with the enzyme was incubated for about 5 minutes at about 37°C in an incubator. After incubation, the flask was gently tapped to dislodge the cells. The contents of the flask is diluted with about 2 ml of Chang's complete media to rinse and stop the reaction. Wash cells with an additional 2 to 5ml DPBS and transfer resuspended cells to a 50 ml centrifuge tube for washing. The tube is centrifuged at 120g for 5 minutes. The supernatant is discarded after centrifugation, and the harvested cells were suspended in Chang's complete media. Use 7ml for a T25 and 15 ml for a T75. See Below for Media: Chang's complete media for Cell Culture, 325 ml of MEM alpha media (Gibco #12571- 063), 90 ml of Chang B (basal media) (18% v/v) (Irvine Scientific, CI 10), 10 ml of Chang C (2% v/v) (Irvine Scientific, Supplement CI 06) , 5 ml Penicillin/Streptomycin (10,000 units/ml Penicillin G Sodium & 10,000 ug/ml Streptomycin sulfate (Gibco #15140-122), 5 ml of L-glutamine 200 mM (100X) (Gibco, #25030-081) and 75 ml of ES-Fetal Bovine Serum (15% v/v) (Gibco #10439-024). [0066] A flow cytometry analysis of a fresh or thawed post-processing sample may be performed by spinning the mixture of the heterogeneous population of cells and cryopreservation agent in a vial in a centrifuge at about 2000 RPM for about 7 minutes between about 15°C to about 30°C until complete or alternatively between about 2°C to about 30°C. After centrifugation, the supernatant is pipetted off and the pellet is diluted in a wash solution. As an example, wash solution may comprise HBSS (Hanks with Ca+ and Mg+) 100ml, Heparin 1 mL or 1000 units, Human Serum Albumin 25% 10 mL and DNASE - 10 drops (Pulmozyme).

[0067] If the heterogeneous population of cells were frozen and need to be thawed the vials should be agitated in a 37°C waterbath and mixed by inversion while avoiding a complete thaw. The quality control will be performed to assess the total number of cells, the number of cells expressing the cell surface marker CDl 17, and the cell viability using 7AAD. Cells expressing the cell surface marker CDl 17 may be assessed by flow cytometry using a monoclonal antibody against CDl 17 with a fluorescent label. For example, suitable monoclonal antibodies include, but are not limited to, BD Pharmingen PE antihuman CDl 17 (YB5.B8) and CD117-PE (104D2D1) or CD1 17-PE (95C3) both from Beckman Coulter.

[0068] Data relating to cryopreserved stem cells obtained from lochia and later thawed according to the present invention is attached hereto as Table B.

[0069] Table B

M38107RA 59,750,000 76.82% 5,215,000 43.51% 17.46% 39,015,000 67.09% 130.59%

M38112RA 16,795,000 36.18% 2,948,000 22.45% 35.11% 14,135,000 41.25% 168.32%

[0070] Accurately pipette approximately between about 0.5 million cells to about 10.0 million cells of a well-mixed sample of the heterogeneous population of cells into two tubes and vortex briefly.

[0071] Wash the cells in the two tubes with about one ml of wash solution and centrifuge in Blood Bank Serofuge for about 1 minute. Decant supernatant without disturbing the pellet. Add about 100 uL of wash solution with a micropipetter back into each tube. Vortex briefly. In the tube for testing, add from about 5ul to about 10 uL of CDl 17-PE dependent upon assay validation, about 20 uL of CD44-FITC, about 10 uL of CD45-ECD, and about 20 uL of 7-AAD Viability dye. In the tube for isotype control, add about 5uL to about 10 uL of IgG-PE dependent upon assay validation, about 20 uL of IgG-FITC, about 10 uL of IgG-EDC and about 20 uL of 7-AAD Viability dye. Incubate the tubes at room temperature (between about 15°C to about 30°C) for about 20 minutes while protecting from light exposure. If sample contains red blood cells with a hematocrit of greater than 5%, lyse for about ten minutes and protect from light. However, if sample was collected by density gradient or obtained from a thawed sample, do not lyse sample. Program carousel work list on FC500 instrument or equivalent. If sample was not lysed, wash after about 20 minutes incubation with about one ml of wash solution and decant supernatant. If sample was lysed, serofuge sample and decant supernatant. Add wash solution up to about one ml and spin again and decant supernatant. Add about one ml of Sheath fluid, vortex and run on FC500 or other suitable flow cytometer. [0072] The CD 117 positive cell count and cell viability are reported from the flow cytometry reports and transcribed to the work documents for thawed post-processing samples. The post sample includes the isotype control result which will be subtracted, if applicable, from the total post CD 1 17+ count and documented on the CD 117+ post report.

[0073] The CD 117 stem cells may be separated from a cellular suspension in working buffer using a MS column to separate the CD1 17 stem cells. For example, an MS Column (Miltenyi Biotec) or other suitable column may be used. Alternatively, other suitable methods to separate cells may be used. A MiniMACS kit available through Miltenyi Biotec comprising a unit, multistand, MS columns and microbeads may be used for CD 1 17 cell selection. The MS column may be prepared by rinsing it with working buffer. For example, the volume of working buffer used to rinse the column may be about 500 ul. The column is placed in a magnetic field of a MACS separator available through Miltenyi Biotec or suitable separator providing a magnetic field.

[0074] The lochia cells may be separated from a cellular suspension by using a commercially available system manufactured by Stem Cell Technologies to perform a negative selection aimed at enrichment of the mesenchymal stem cells in the sample preparation. A cocktail with monoclonal antibodies to the following human cell surface antigens: Glycophorin A, CD3, CD 14, CD 19, CD66b, CD38 may be used to increase target cells 8- to 10-fold enrichment of mesenchymal stem cells.

[0075] The heterogeneous population of cells collected in accordance with the methodologies of the present study may be cultured. The heterogeneous population of cells may be prepared for cell culture after concentration according to the present study or after being cryopreserved and thawed. When applicable, the thawing step comprises preparing aliquots of about 15 ml of density gradient media available as Histopaque through Sigma-Aldrich or other suitable media at about room temperature for each vial containing about 5 ml of cryopreserved cells to be thawed; and then preparing about 25 ml aliquots of Chang's complete media, DMEM complete media or IMDM complete media or other suitable media for each vial containing about 5 ml of cryopreserved cells to be thawed.

[0076] Chang's complete media comprises about 325 ml of MEM alpha media available through Gibco as product 12571-063, Add 20% Chang's Media to include about 90 ml of Chang B (basal) CI 10 (18% v/v) available through Irvine Scientific, about 10 ml of Chang medium C from Supplement CI 06 (2% v/v) available through Irvine Scientific, about 5 ml Penicillin/Streptomycin (liquid prepared with 10,000 units/ml Penicillin G Sodium and 10,000 ug/ml Streptomycin sulfate in 0.85% saline available through Gibco as product 15140-122, about 5 ml of L-glutamine 200 raM (100X) available through Gibco as product 25030-081, and about 75 ml of ES-Fetal Bovine Serum (15% v/v) available through Gibco as product 10439-024.

[0077] If thawing of a cryopreserved sample is necessary, in an embodiment, the cryopreserved cells are thawed by removing the vials from the vapor phase of the liquid nitrogen storage freezer. The vials are placed in about a 37°C to 40°C water bath and agitated. The cells should not be allowed to completely thaw, but the vials should contain ice. The thawed cells should be diluted by placing the about 5 ml aliquot into the about 25 ml aliquot of chilled Chang's complete media containing about one mg of DNAse available through Pulmozyme. Alternatively, and if thawing is not necessary, such as for example, when the heterogeneous population of cells is cultured in the absence of the step of cryopreservation, the cellular suspension should be diluted by placing the about 5 ml aliquot into the about 25 ml aliquot of chilled Chang's complete media containing about one mg of DNAse available through Pulmozyme.

[0078] The diluted cell suspension may be mixed by inversion. The suspension is centrifuged at about 840 g for about 7 minutes. The supernatant is aspirated while not disturbing the pellet. The pellet is brought up to a total volume of about 30 ml Chang's complete media. A small amount of the cell suspension is removed for analysis that includes cell count with a hemocytometer and viability testing using trypan blue or other suitable viability testing methodology. The about 30 ml suspension is overlaid on a density gradient solution available as Histopaque through Sigma-Aldrich or other suitable media, and is centrifuged at about 420 g for about 30 minutes without a brake. The tube is removed from the centrifuge without disrupting the buffy coat. The supernatant is aspirated and the buffy coat is collected. The buffy coat is brought up to about 20 ml with Chang's complete media and is washed at about 840 g for about 7 minutes. The supernatant is aspirated, and the pellet is suspended in Chang's complete media up to about 10 ml but may also up to about 20 ml or about 30 ml, or even less than about 10 ml. An aliquot of the suspension, such as for example, about 100 ul, is removed to perform a cell count and viability analysis.

[0079] The cells in suspension may be seeded at about 40,000 cells/cm into an untreated culture flask in Chang's complete media, DMEM complete media (with high glucose or low glucose), or other suitable media. The flask should be incubated in about 5% C0₂ in a C02 incubator available through Thermo Electron Corp. or Bioscience Technologies, or any other suitable incubator system at a temperature of about 37°C. The cell cultures are monitored for turbidity and pH change. If the pH is high, about 50% of the media should be changed.

[0080] The flask may be incubated initially for about 7 days or until the media is significantly out of range as noted by the color of the phenol red indicator in the media. If the pH remains stable after about 7 days, the media is changed with fresh media (also referred to herein as "virgin media"), as necessary. Almost all media is changed. After the media change at day 7, the cells may become confluent by day 8 to day 21. Once attaining about 70-80% confluence, the cells may be sub-cultured. Cell cultures are sub- cultured using the trypsin-like enzyme such as TrypLE™ Express available through Gibco, or any other suitable enzyme to provide enough cells to perform the CD117 cell selection in accordance with the present study.

[0081] For example, cell selection may occur with about 10 million cells. Cell selection may also occur with greater than or less than about 10 million cells. In accordance with the study, CD1 17 cells may be collected from the cell culture at a suitable time. In order to collect the CD117 cells, adherent cells should be dissociated from the flask. In order to dissociate the cells from the flask, the media is aspirated via an automated pipette. The flask is then rinsed with about 5 ml of Phosphate Buffered Saline (PBS) without calcium or magnesium. The PBS is then removed from the flask with attached cells that have been washed at least once. About one ml of a Trypsin-like recombinant enzyme such as TrypLE™ Express available through Gibco, or any other suitable enzyme, should be added, preferably pre- warmed at about 37°C, to the cell culture in the flask. The flask is agitated to coat the cells with the enzyme. The flask with enzyme should be incubated for about 5 minutes at about 37°C. After incubation, the flask should be gently tapped on a solid surface to dislodge the cells. The flask should be diluted with about 2 ml of Chang's complete media, and the cells transferred to a 15 ml centrifuge tube for washing with Chang's complete media, DMEM complete media (with high glucose or low glucose), or other suitable media. The tube should be centrifuged for about 7 minutes at about 100 x g. The supernatant is aspirated and discarded. The pellet is suspended in a suitable volume of Chang's complete media, DMEM complete media (with high glucose or low glucose), or other suitable media.

[0082] At this point, the CD117 cells may be selected from the cell culture in accordance with CD117 cell selection methodologies of the present study or a negative selection may be performed. The cells may be plated in a 9cm Petri dish using Chang's complete media (about 15% FBS). Alternatively, the cells may be placed in a culture flask with a vented cap. If the pH of the media becomes high, the cells may be washed with Chang's complete media. When necessary after suitable growth, the cells may be dissociated from the Petri dish or culture flask using a trypsin-like enzyme and then placed in an untreated culture flask using Chang's complete media. After suitable growth, the cells may be dissociated using a trypsin-like enzyme such as TrypLE™ Express available through Gibco and then seeded in a fresh untreated culture flask. This process may be repeated in order to maintain desired cell growth. The cells may be washed with fresh media, or about 50% of the media or other suitable amount may be replaced with fresh media if the pH of the media is high. At this point, the CD117 cells may be selected from the cell culture in accordance with the CD117 cell selection methodology of the present study. The selected CD 1 17 cells may be plated on a Petri dish, seeded into a culture flask or cryopreserved.

[0083] Therapeutic Uses

[0084] The lochia cells of the present invention may be prepared by combining the cells with a suitable cell media or pharmaceutically acceptable excipient. For example, the lochia cells of the invention may be prepared for infusion or transplant according to GMP (Good Manufacturing Practices). Suitable media or diluents may include tissue culture media (including proper testing and results documented on a certificate of analysis), such as, HBSS, IMDM, RPMI, Alpha MEM, Ml 99, and DMEM, which may require HEPES buffer to maintain an appropriate pH to sustain the menstrual stem cells. Protein may be added at minimum of about 0.2%, but may be added as high as about 30%. Protein used may also include Human Serum Albumin, Human Plasma Protein Fraction, Fetal Calf Serum (if using animal products they must include proper safety testing as required by the United States Food and Drug Administration), or Bovine Serum Albumin. The media may require an anticoagulant if the cells are at risk of coagulation, clumping, or clot. Anticoagulants may include Heparin (preservative free is preferred as preservative may potentially affect the stem cell growth and proliferative capability of the cells), Acid Citrate Dextrose, Citrate Phosphate Dextrose, or any suitable anticoagulant that is safe to combine with viable cells may be used. Cells may be shipped in the cell media they are expanded in as long as it is safe for infusion or transplant.

[0085] Lochia cells may be shipped in a syringe for a short distance, for example, if the product is being prepared in the hospital and also administered in the hospital. Otherwise products may be transported in sterile bags, which may or may not include gas exchange. Products shipped short distance may also be transported in any other type of sterile container that the cells will tolerate and remain viable in for the short distance, which may include a sterile conical tube, or a sterile flat bottom container, which may be round or square. If the product is being shipped long distance more than 4 hours it may be sent in a bag that allows for gas exchange. The bag may also be shipped in a configuration that allows for air exchange. The product bag may be placed on a rack that will allow for proper gas exchange in the container it is being shipped in. The container including the cells may be optimized for a suitable temperature to maintain the menstrual stem cells. If the cells require a cool environment, ice packs or other cooling device may be used. If the cells require room temperature at about 20.degree. C. to about 24.degree. C, then a gel pack may be required to maintain this temperature.

[0086] Once the lochia cells arrive at the facility for infusion or transplant, the lochia cells may be placed in a monitored temperature environment. Lochia cells that will be stored for use at a later date may be shipped cryopreserved in a dry shipper to maintain temperatures less than or equal to -150.degree. C. Lochia cells shipped cryopreserved may be sent with thaw and infusion instructions and potentially the media required to maintain an optimally viable cell product for therapeutic use.

[0087] A suitable transport mechanism should be used to transport the product to the infusion site. Any number of different types of transport services may be used, such as, for example, a medical courier service, a courier service, or a company such as UPS, DHL, or FedEx. The lochia cells in media have a greater chance of safe transportation when the courier understands the products needs such as the fact that a Dry Shipper must remain upright for the duration of travel. If the shipper is placed on its side it, may quickly loss the charge of liquid nitrogen and fail to hold the temperature it was validated for.

[0088] Cosmeceutical Applications

[0089] The term cosmeceutical is used in reference to a formulation or composition comprising (a) at least one biologically active ingredient that have an effect on the user of the product, and (b) at least one cosmeceutically-acceptable carrier. The biologically active ingredient may be, for example, lochia cells collected in accordance with the practice of this invention, or growth factors or other compositions, chemicals, or proteins produced by lochia cells, genetically-engineered lochia cells, or differentiated lochia cells of the present invention. By way of further example, lochia cells, differentiated lochia cells, or genetically-engineered lochia cells may be grown in culture to produced a desired cellular product, such as, for example, a growth factor or other biologically active ingredient capable of use as a cosmeceutical. The growth factor or other biologically active ingredient may be collected from cell culture media or other media that the lochia cells, differentiated lochia cells, or genetically-engineered lochia cells are grown. The growth factor or other biologically active ingredient may be collected from cell culture media or other media by way of centrifugation steps to collected the desired ingredient in a supernatant, which may be later processed to purify or concentrated the ingredient.

[0090] Cosmeceuticals may be viewed as cosmetics that, in certain applications and under appropriate conditions, may provide medicinal or drug-like benefits. In certain applications, for example, cosmeceuticals may affect the underlying structure of the skin, decrease wrinkle depth, or reverse or ameliorate the effect of photooxidation or aging on the skin. Cosmeceuticals may be particularly useful as skin care products, hair care products, tissue or organ modification or augmentation, and sun care products. In certain embodiments, cosmeceutical compositions comprise delivery systems including at least one of liposomes, cyclodextrins, polymer systems, or hyaluronic acid or related compounds. Cosmeceutical compositions comprise cosmeceutically-acceptable carriers. A pharmaceutically-acceptable carrier or formulation that is suitable for topical applications will typically also be a cosmeceutically-acceptable carrier or formulation.

[0091] A topical cosmetic or cosmeceutical ointment, lotion, or gel composition typically contains a concentration of active ingredients comprising conditioned media or extracts thereof, from about 1 to 99%, about 5 to 95%, about 20 to 75%, or about 5 to 20%, in a cosmetically-acceptable carrier or a cosmeceutically-acceptable carrier, such as a pharmaceutical cream base, an oil-in-water emulsion, a water-in-oil emulsion, a gel, or the like. Various cosmetic and cosmeceutical compositions for topical use include drops, tinctures, lotions, creams, salves, serums, solutions, and ointments containing conditioned media or extracts, and an appropriate carrier. The optimal percentage of the conditioned media or extract in each composition varies according to the composition's formulation and the therapeutic effect desired.

[0092] The cosmoceuticals may comprise any of a number of cosmetically-, cosmeceutically, or pharmaceutically-acceptable formulations, depending on the type of product, the nature of the composition, the location of composition's use, the desired effect, and the like. While proprietary formulations are common in the formulation arts, formulators will be able to determine or readily select appropriate formulations for specific applications without undue experimentation. [0093] Appropriate carriers of the inventive compositions typically will contain ingredients, such as those typically found in the cosmetic and cosmeceutical fields: oils, waxes or other standard fatty substances, or conventional gelling agents and/or thickeners; emulsifiers; moisturizing agents; emollients; sunscreens; hydrophilic or lipophilic active agents, such as ceramides; agents for combatting free radicals; bactericides; sequestering agents; preservatives; basifying or acidifying agents; fragrances; surfactants; fillers; natural products or extracts of natural product, such as aloe or green tea extract; vitamins; or coloring materials. The amounts of these various ingredients will vary depending on the use of the composition and the cosmetic or cosmeceutical effect desired.

[0094] Discussions of cosmetic- and cosmeceutically-acceptable ingredients and formulations may be found in, among other places, FDA Cosmetics Handbook, U.S. Food and Drug Administration; Handbook of Cosmetic and Personal Care Additives, Ash and Ash, compilers, 1994, Chemical Publishing, New York, N.Y.; Bennett's Cosmetic Formulary, 1993, Chemical Publishing Co.; Harry's Cosmeticology, 7.sup.th ed., Wilkinson & Moore, 1982 and 8.sup.th ed., Rieger, 2000, Chemical Publishing; Cosmetic Bench Reference-2001, Allerud Publishing Corp.; CTFA Compendium of Cosmetic Ingredient Composition, Nikitakis and McEwen, eds., 1990, Cosmetic, Toiletry, and Fragrance Association, Washington, D.C., Surfactant Encyclopedia, 2.sup.nd revised edition, Rieger, 1996, Allured Publishing; The Chemistry and Manufacture of Cosmetics, 2.sup.nd ed., De Navarre, Van Nostrand, Princeton, N.J.; Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics, Leung, 1996, John Wiley; A Consumer's Dictionary of Cosmetic Ingredients, 5.sup.th ed., Winter, 1999, Three Rivers Press, New York, N.Y.; Cosmeceuticals: Active Skin Treatment, 1998, Allured Publishing; Handbook of Cosmetic Science and Technology, Knowlton and Pearce, 1993, Elsevier Advanced Technology, Oxford, UK; Personal-Care Formulas, 1997, Allured Publishing; Beginning Cosmetic Chemistry, Scheuller and Romanowski, 1999, Allured Publishing; and Skin Permeation: Fundamentals and Application, Zatz, 1993, Allured Publishing. Discussions of pharmaceutically-acceptable ingredients and formulations may be found in, among other places, Remington's Pharmaceutical Sciences, 18.sup.th ed., Gennaro, ed., 1990, Mack Publishing.

[0095] While preferred embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that many changes and modifications may be made without departing from the invention in its broader aspects. The appended claims are intended to cover, therefore, all such changes and modifications as fall within the true spirit and scope of the invention.

Claims

The invention claimed is:

1. A method for obtaining stem-like cells from lochia, the method comprising the steps of:

isolating a population of cells from the lochia; and

selecting stem-like cells from the isolated population of cells.

2. The method of Claim 1, wherein the method comprises cryopreserving the selected stem-like cells.

3. The method of Claim 2, wherein the method comprises thawing the cryopreserved stem-like cells for therapeutic, research, diagnostic, or medical use.

4. The method of Claim 3, wherein the method comprises combining the thawed stem-like cells with a pharmaceutically-acceptable excipient, a cosmeceutical agent, a therapeutic agent, or a media.

5. The method of Claim 1, wherein the method comprises expanding the stem-like cells in culture.

6. The method of Claim 5, wherein the method comprises cryopreserving the expanded stem-like cells.

7. The method of Claim 1, wherein the method comprises combining the selected stem-like cells with a pharmaceutically-acceptable excipient, a cosmeceutical agent, a therapeutic agent, or a media.

8. The method of Claim 1, wherein the step of selecting stem-like cells comprises positively selecting cells for one or more of CD29, CD44, CD49e, CD54, CD73, CD90, CD105, CD117, CD133, CD166, HLA class I, SSEA-3, SSEA-4, Oct-4, and nanog.

9. The method of Claim 1, wherein the step of selecting stem-like cells comprises negatively selecting cells for one or more of CD 14, CD31, CD34, CD45 and HLA class II.

10. A method for isolating viable stem-like cells from lochia, the method comprising the steps of:

collecting a sufficient amount of lochia;

suspending the lochia in a stabilization media;

removing a population of cells from the suspended lochia; and isolating the viable stem-like cells from the suspended lochia.

11. The method of Claim 10, wherein the method comprises isolating the viable stem-like cells within about 24 to about 72 hours from collecting the sufficient amount of lochia.

12. The method of Claim 10, wherein the method comprises cryopreserving the selected stem-like cells.

13. The method of Claim 12, wherein the method comprises thawing the cryopreserved stem-like cells for therapeutic, research, diagnostic, or medical use.

14. The method of Claim 10, wherein the method comprises expanding the stem-like cells in culture.

15. The method of Claim 14, wherein the method comprises expanding the stem-like cells in culture.

16. The method of claim 10, wherein the step of selecting stem-like cells comprises positively selecting cells for one or more of CD29, CD44, CD49e, CD54, CD73, CD90, CD105, CD1 17, CD133, CD166, HLA class I, SSEA-3, SSEA-4, Oct-4, and nanog.

17. The method of Claim 10, wherein the step of selecting stem-like cells comprises negatively selecting cells for one or more of CD 14, CD31, CD34, CD45 and HLA class II.

18. A composition of matter comprising lochia cells and any one of a pharmaceutically-acceptable excipient, cosmeceutical agent, therapeutic agent, or media.