Patent Application
20060183102
The present invention relates generally to serum-free reagents suitable for the isolation, cultivation, and cryopreservation of lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSC). The present invention further relates to methods for making and using the serum-free reagents and stem cells associated therewith.
The growth medium for cells grown in culture is routinely supplemented with animal and/or human serum to optimize and enhance cell viability. The constituents of serum include water, amino acids, minerals, vitamins, fatty acids, triglycerides, monosaccharides, disaccharides, polysaccharides, proteins, glycoproteins, glycolipids, proteoglycans, glycosaminoglycans, etc. Either singly or in combination, these serum constituents act as bioactive agents by impacting on the functional capabilities of the cells grown in culture. Potential bioactive agents present in serum include agents that induce proliferation, agents that induce differentiation, agents that accelerate phenotypic expression, agents that inhibit proliferation, agents that inhibit differentiation, and/or agents that inhibit phenotypic expression. Unfortunately, the identity(ies), concentration(s), and potential combinations of specific bioactive agents contained in different lots of animal or human serum is/are unknown. One or more of these unknown agents in serum have shown to have a negative impact on the isolation, cultivation, and cryopreservation of lineage-uncommitted postnatal pluripotent epiblast-like stem cells.
To circumvent the problems inherent with the use of animal and/or human serum in culture medium, serum-free solutions have been devised for the isolation, cultivation, and cryopreservation, of lineage-uncommitted postnatal pluripotent epiblast-like stem cells.
The present invention is directed to a series of serum-free solutions suitable for use with postnatal pluripotent epiblast-like stem cells. The serum-free solutions may be used in the isolation, cultivation, and cryopreservation of lineage-uncommitted postnatal pluripotent epiblast-like stem cells.
According to one exemplary embodiment of the present invention, a serum-free defined solution to wash tissues prior to the tissue harvest of postnatal pluripotent epiblast-like stem cells comprises (a) a filter sterilized buffer solution comprising (i) Dulbecco's phosphate buffered saline containing calcium chloride and magnesium chloride, (ii) albumin, (iii) 2-mercaptoethanol, (iv) putrescine, (v) an antibiotic-antimycotic solution; and (vi) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In a further exemplary embodiment of the present invention, a serum-free defined tissue harvest and storage medium for postnatal pluripotent epiblast-like stem cells comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, trace elements and growth factors (i.e., OPTI-MEM I); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; (f) collagen, and (g) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined cell medium for the isolation of postnatal pluripotent epiblast-like stem cells from body tissues comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, trace elements and growth factors (i.e., OPTI-MEM I); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; (f) dispase, (g) collagenase, and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined plating medium for postnatal pluripotent epiblast-like stem cells comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, trace elements and growth factors (i.e., OPTI-MEM I); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; (f) collagen, (g) fibronectin, (h) platelet-derived growth factor-BB, and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In a further exemplary embodiment of the present invention, a serum-free defined cell wash solution for cultured postnatal pluripotent epiblast-like stem cells comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, GLUTAMAX, trace elements and growth factors (i.e., OPTI-MEM I with GLUTAMAX); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined propagation medium for postnatal pluripotent epiblast-like stem cells comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, trace elements and growth factors (i.e., OPTI-MEM I); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; (f) collagen, (g) fibronectin, (h) platelet-derived growth factor-BB, and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined release solution-A for releasing postnatal pluripotent epiblast-like stem cells from culture surfaces comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) containing L-glutamine and buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, trace elements and growth factors (i.e., OPTI-MEM I with GLUTAMAX); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined release solution-B for releasing postnatal pluripotent epiblast-like stem cells from culture surfaces comprises (a) Dulbecco's phosphate buffered saline optionally containing calcium chloride and magnesium chloride; (b) an optional antibiotic-antimycotic solution; (c) a putrescine-containing solution; (d) 2-mercaptoethanol solution; and (e) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined release solution-C for releasing postnatal pluripotent epiblast-like stem cells from culture surfaces comprises (a) Dulbecco's phosphate buffered saline without calcium chloride and without magnesium chloride; (b) an optional antibiotic-antimycotic solution; (c) a putrescine-containing solution; (d) 2-mercaptoethanol solution; (e) an EDTA solution; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined release solution-D for releasing postnatal pluripotent epiblast-like stem cells from culture surfaces comprises (a) Dulbecco's phosphate buffered saline without calcium chloride and without magnesium chloride; (b) an optional antibiotic-antimycotic solution; (c) a putrescine-containing solution; (d) 2-mercaptoethanol solution; (e) an EDTA solution; (f) trypsin, and (g) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a serum-free defined cryopreservation medium for postnatal pluripotent epiblast-like stem cells comprises (a) a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, trace elements and growth factors (i.e., OPTI-MEM I); (b) an optional antibiotic-antimycotic solution; (c) putrescine; (d) 2-mercaptoethanol solution; (e) albumin; (f) collagen, (g) fibronectin, (h) platelet-derived growth factor-BB, (i) dimethylsulfoxide and (j) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
The present invention is directed to serum-free solutions suitable for use with postnatal pluripotent epiblast-like stem cells. The serum-free solutions may be used in the isolation, cultivation, and cryopreservation of lineage-uncommitted postnatal pluripotent epiblast-like stem cells. The present invention is further directed to methods of using serum-free solutions in the isolation, cultivation, and cryopreservation of lineage-uncommitted postnatal pluripotent epiblast-like stem cells.
I. Serum-Free Solutions
The present invention is directed to a variety of serum-free solutions suitable for use with postnatal pluripotent epiblast-like stem cells. In one exemplary embodiment of the present invention, the serum-free solution comprises (a) a filter sterilized buffer solution comprising (i) (i) Dulbecco's phosphate buffered saline containing calcium chloride and magnesium chloride, (ii) albumin, (iii) 2-mercaptoethanol, (iv) putrescine, (v) an antibiotic-antimycotic solution (described below); and an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. This exemplary serum-free solution is particularly suitable for washing tissue prior to tissue harvest of postnatal pluripotent epiblast-like stem cells.
The exemplary serum-free solution contains Dulbecco's phosphate buffered saline containing calcium chloride and magnesium chloride. (also referred to herein as “a buffered saline solution”). The composition of Dulbecco's phosphate buffered saline containing calcium chloride and magnesium chloride is shown in Table 1 below.
The serum-free solution further comprises an antibiotic-antimycotic solution. Suitable antibiotic-antimycotic solutions comprise, but are not limited to, antibiotic-antimycotic solutions containing at least one of penicillin, streptomycin, and fungizone. Desirably, the 100X antibiotic-antimycotic solution comprises about 10,000 units/ml Penicillin G, about 10,000 μg/ml Streptomycin, and about 25 μg/ml Amphotericin B, more desirably, a preferred antibiotic-antimycotic solution comprises 10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B (hereinafter referred to as “the preferred antibiotic-antimycotic solution”). Such antibiotic-antimycotic solutions are commercially available from Mediatech, Inc. (Herndon, Va.) under the trade designation CELLGRO® and commercially available from Invitrogen Corporation (Carlsbad, Calif.) under the trade designation GIBCO®.
Other suitable antibiotic-antimycotic solutions include, but are not limited to, solutions containing about 10,000 units/ml Penicillin G and/or about 10,000 μg/ml Streptomycin and/or about 25 μg/ml Amphotericin B; or any other type of antibiotic and/or antimycotic solution capable of preventing microbiological contamination of the cultured cells. Such other suitable antibiotic-antimycotic solutions are also commercially available from Mediatech, Inc. (Herndon, Va.) and Invitrogen Corporation (Carlsbad, Calif.).
The putrescine-containing solution may comprise a filter sterilized solution containing putrescine and one or more additional components. In one exemplary embodiment, the putrescine-containing solution comprises a filter sterilized solution containing putrescine, cell culture media, and an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a putrescine-containing solution having a pH of 7.4. The cell culture media desirably comprises OPTI-MEM I with GLUTAMAX commercially available from Gibco/Invitrogen Corporation (Carlsbad, Calif.). OPTI-MEM I with GLUTAMAX comprises a modification of Eagle's Minimal Essential Medium buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, GLUTAMAX, trace elements and growth factors (hereinafter referred to as “the second preferred cell culture solution”).
The above-described serum-free solution desirably comprises (a) greater than about 90 percent by weight of the filter sterilized buffer solution, wherein the filter sterilized buffer solution comprises (i) greater than about 90 percent by weight Dulbecco's phosphate buffered saline containing calcium chloride and magnesium chloride, (ii) up to about 5.0 percent by weight albumin, (iii) greater than 0 up to about 6.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (iv) greater than 0 up to about 1.0 percent by weight of a putrescine-containing solution; (v) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; and (vi) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In a further desired embodiment, the above-described serum-free solution comprises (a) 486 ml of the filter sterilized buffer solution, wherein the filter sterilized buffer solution comprises (i) Dulbecco's phosphate buffered saline containing calcium chloride and magnesium chloride, (ii) 0.5 percent by weight of albumin, (iii) 15 ml of the preferred antibiotic-antimycotic solution; (a) 0.1 ml of a 1M putrescine solution comprising (i) 1.61 g of putrescine; (ii) 10 ml of OPTI-MEM I with GLUTAMAX (i.e., “the second preferred cell culture solution”); and (iii) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a putrescine-containing solution having a pH of 7.4; (b) 0.9 ml of a 55 mM 2-mercaptoethanol solution; and (c) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
The present invention is further directed to serum-free solutions suitable for use as a tissue harvest and storage medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSC). One such serum-free solution comprises (a) greater than 90 percent by weight of a cell culture solution (i.e., Eagle's Minimal Essential Medium) buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, phenol red, trace elements and growth factors (i.e., OPTI-MEM I) (hereinafter referred to as “the first preferred cell culture solution”); (b) greater than 0 up to about 6.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 10.0 percent by weight of albumin; (f) greater than 0 up to about 1.0 percent by weight of type-I collagen; (g) up to about 1.0 percent by weight of optional fibronectin; and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In a specific solution suitable for use for diagnostic purposes, the serum-free tissue harvest and storage solution comprises (a) 484.4 ml of the first preferred cell culture solution; (b) 15.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 5.0 g of serum albumin; (f) 50 mg of type-I collagen; and (g) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In a further embodiment, the serum-free solution suitable for use as a tissue harvest and storage medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSC) comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the first preferred cell culture solution; (b) greater than 0 up to about 6.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 10.0 percent by weight of albumin; (f) greater than 0 up to about 1.0 percent by weight of human type-I collagen; (g) greater than 0 up to about 1.0 percent by weight of fibronectin; and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In a specific embodiment for human clinical purposes, the serum-free tissue harvest and storage solution suitable for use as a tissue harvest and storage medium comprises (a) 484.4 ml of the first preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 5.0 g of the human serum albumin; (f) 50 mg of human type-I collagen; and (g) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
The present invention is further directed to serum-free solutions suitable for use as a cell isolation medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSCs). One such serum-free solution comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the first preferred cell culture solution; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 3.0 percent by weight of albumin; (f) up to about 0.6 g of type-I collagenase; (g) about 50 ml of a dispase solution (described below); and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In a specific solution suitable for use for diagnostic purposes, the serum-free cell isolation solution comprises (a) 484.4 ml of the first preferred cell culture solution; (b) 15.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 5.0 g of serum albumin; (f) 250 units per ml of collagenase; (g) 33.3 units of dispase per ml of serum-free solution; and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In a further embodiment, the serum-free solution suitable as a cell isolation medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSC) comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the first preferred cell culture solution; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 3.0 percent by weight of albumin; (f) up to about 500 units per ml of type-I collagenase; (g) up to about 70 units per ml of a dispase solution, and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In a specific embodiment for human clinical purposes, the serum-free cell isolation solution suitable for use as a tissue harvest and storage medium comprises (a) 484.4 ml of the first preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 5.0 g of human serum albumin; (f) 250 units per ml of collagenase; (g) 33.3 units of dispase per ml of serum-free solution; and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
The present invention is further directed to serum-free solutions suitable for use as a plating medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSCs). One such serum-free solution comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the first preferred cell culture solution; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 10.0 percent by weight of albumin; (f) greater than 0 up to about 5.0 percent by weight of type-I collagen; (g) greater than 0 up to about 1.0 percent by weight of fibronectin; (h) greater than 0 up to about 20.0 ng/ml platelet-derived growth factor-BB (R&D Systems, Minneapolis Minn.); and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free plating solution comprises (a) 494.4 ml of the first preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 5.0 g of albumin; (f) 5.0 g of type-I collagen; (g) 37.5 μg of fibronectin; (h) 5.0 ng/ml platelet-derived growth factor-BB; and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free plating solution comprises (a) 499.4 ml of the first preferred cell culture solution; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (d) 5.0 g of albumin; (e) 5.0 g of type-I collagen; (f) 37.5 μg of fibronectin; (g) 5.0 ng/ml platelet-derived growth factor-BB; and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
The present invention is further directed to serum-free solutions suitable for use as a propagation medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSCs). One such serum-free solution comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the first preferred cell culture solution; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 10.0 percent by weight of albumin; (f) greater than 0 up to about 1.0 percent by weight of type-I collagen; (g) greater than 0 up to about 1.0 percent by weight of fibronectin; (h) greater than 0 up to about 20.0 ng/ml platelet-derived growth factor-BB (R&D Systems, Minneapolis Minn.); and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free propagation solution comprises (a) 494.4 ml of the first preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 1.0 g of albumin; (f) 50 mg of type-I collagen; (g) 37.5 μg of fibronectin; (h) 10.0 ng/ml platelet-derived growth factor-BB; and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free propagation solution comprises (a) 499.4 ml of the first preferred cell culture solution; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (d) 1.0 g of albumin; (e) 50 mg of type-I collagen; (f) 37.5 μg of fibronectin; (g) 10.0 ng/ml platelet-derived growth factor-BB; and (h) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
The present invention is even further directed to serum-free solutions suitable for use as a cell washing medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSCs). One such serum-free solution comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the second preferred cell culture solution (i.e., OPTI-MEM I with GLUTAMAX); (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 10.0 percent by weight of albumin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free cell washing solution comprises (a) 494.4 ml of the second preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 1.0 g of bovine serum albumin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free cell washing solution comprises (a) 499.4 ml of the second preferred cell culture solution; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (d) 1.0 g of human serum albumin; and (e) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In yet a further exemplary embodiment of the present invention, a series of four serum-free defined release solutions are necessary for releasing postnatal pluripotent epiblast-like stem cells from culture surfaces. These serum-free defined solutions, to be used in sequence, are designated release solution-A, release solution-B, release solution-C, and release solution-D.
Release solution-A comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the second preferred cell culture solution; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 20.0 percent by weight of albumin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free defined release solution-A comprises (a) 494.4 ml of the second preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 10.0 g of serum albumin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free defined release solution-A comprises (a) 499.4 ml of the second preferred cell culture solution; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (d) 10.0 g of human serum albumin; and (e) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
Release solution-B comprises (a) greater than 90 percent by weight of the Dulbecco's phosphate buffered saline optionally containing calcium chloride and magnesium chloride; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; and (e) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free defined release solution-B comprises (a) 494.4 ml of the Dulbecco's solution containing calcium chloride and magnesium chloride; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; and (e) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free defined release solution-B comprises (a) 499.4 ml of the Dulbecco's solution containing calcium chloride and magnesium chloride; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; and (d) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
Release solution-C comprises (a) greater than 90 percent by weight of the Dulbecco's phosphate buffered saline without calcium chloride and without magnesium chloride; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to 5.0 ml of a 1.0 M solution of ethylenediamine tetraacetic acid (EDTA); and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free defined release solution-C comprises (a) 492.3 ml of the Dulbecco's phosphate buffered saline solution without calcium chloride and without magnesium chloride; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 2.2 ml of a 0.5 M solution of EDTA; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free defined release solution-C comprises (a) 497.3 ml of the Dulbecco's phosphate buffered saline solution without calcium chloride and without magnesium chloride; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (c) 2.2 ml of a 0.5 M solution of EDTA; and (e) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
Release solution-D comprises (a) greater than 90 percent by weight of the Dulbecco's phosphate buffered saline without calcium chloride and without magnesium chloride; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to 5.0 ml of greater than 0 up to a 1.0 M solution of EDTA; (f) greater than 0 up to 100 mg trypsin; and (g) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free defined release solution-D comprises (a) 492.3 ml of the Dulbecco's phosphate buffered saline solution without calcium chloride and without magnesium chloride; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 2.2 ml of a 0.5 M solution of EDTA; (f) 50 mg trypsin; and (g) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free defined release solution-D comprises (a) 497.3 ml of the Dulbecco's phosphate buffered saline solution without calcium chloride and without magnesium chloride; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (c) 2.2 ml of a 0.5 M solution of EDTA; (e) 50 mg trypsin; and (f) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In the above-described serum-free solutions of the present invention, the albumin used in the compositions may comprise bovine serum albumin to prepare solutions suitable for diagnostic purposes, or human serum albumin to prepare solutions suitable for human clinical purposes.
The present invention is further directed to serum-free solutions suitable for use as a cryopreservation medium for lineage-uncommitted postnatal pluripotent epiblast-like stem cells (PPELSCs). One such serum-free solution comprises (a) greater than 90 percent by weight of a cell culture solution, desirably, the first preferred cell culture solution; (b) greater than 0 up to about 2.0 percent by weight of an antibiotic-antimycotic solution, desirably, the preferred antibiotic-antimycotic solution; (c) greater than 0 up to about 1.0 percent by weight of putrescine; (d) greater than 0 up to about 1.0 percent by weight of 2-mercaptoethanol; (e) greater than 0 up to about 20.0 percent by weight of albumin; (f) greater than 0 up to about 1.0 percent by weight of type-I collagen; (g) greater than 0 up to about 1.0 percent by weight of fibronectin; (h) greater than 0 up to about 20.0 ng/ml platelet-derived growth factor-BB (R&D Systems, Minneapolis Minn.); (i) greater than 0 up to about 500 μl/ml dimethylsulfoxide; and (j) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
In one specific embodiment for diagnostic purposes, the serum-free cryopreservation solution comprises (a) 494.25 ml of the first preferred cell culture solution; (b) 5.0 ml of the preferred antibiotic-antimycotic solution; (c) 0.05 mg of putrescine; (d) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (e) 1.0 g of albumin; (f) 50 mg of type-I collagen; (g) 37.5 μg of fibronectin; (h) 5.0 ng/ml platelet-derived growth factor-BB; (i) 150 μl/ml of 99.999% pure dimethyl sulfoxide; and (j) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4. In another specific embodiment for human clinical purposes, the serum-free cryopreservation solution comprises (a) 499.25 ml of the first preferred cell culture solution; (b) 0.05 mg of putrescine; (c) 0.5 ml of a 55 mM 2-mercaptoethanol solution; (d) 1.0 g of albumin; (e) 50 mg of type-I collagen; (f) 37.5 μg of fibronectin; (g) 5.0 ng/ml platelet-derived growth factor-BB; (h) μl/ml of 99.999% pure dimethyl sulfoxide; and (i) an optional amount of sterile sodium hydroxide or sterile hydrochloric acid to provide a serum-free solution having a pH of 7.4.
II. Methods of Making Specific Serum-Free Solutions
The present invention is further directed to methods of making the above-mentioned serum-free solutions. Methods of making a variety of specific serum-free solutions are provided below.
A. Antibiotic-Antimycotic Solution
In one embodiment, the preferred antibiotic-antimycotic solution comprises 10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B and is commercially available from Mediatech (Cellgro, Herndon, Va.) as a 100X solution. The 100X preferred antibiotic-antimycotic solution is added to serum-free solutions at a range of 0 to 6 mls per 100 ml solution (0 to 30 mls per 500 ml) for a final concentration range of 0 to 6%. Alternate solutions utilized with the cultured cells include similar 100X antibiotic-antimycotic solutions obtained from other companies, e.g., 10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B [GIBCO (Invitrogen Corporation, Carlsbad, Calif.) 100X], or various combinations or single versions of sterile antibiotic(s) and/or antimycotic(s) (from any company) necessary to prevent microbiological contamination of the cultured cells, i.e., 10,000 units/ml Penicillin G and 10,000 μg/ml Streptomycin [either Mediatech or GIBCO]; 10,000 units/ml Penicillin G [either Mediatech or GIBCO]; 10,000 μg/ml Streptomycin [either Mediatech or GIBCO]; and 25 μg/ml Amphotericin B [either Mediatech or GIBCO] alone or in combination with one another.
B. Disinfectant
The disinfectant of choice is Amphyl solution (Reckitt & Coleman Inc., Montvale, N.J.): 0.5% (v/v) in deionized water. In a 20 L carboy, mix 100 ml of Amphyl with 19.9 L of deionized water. Swirl the carboy gently to mix the contents. However, 70% ethanol or other disinfectants not harmful to the cells may be utilized.
C. 70% (v/v) Ethanol
Dilute 95% ethanol (Fisher Scientific, Pittsburgh, Pa.) to 70% (v/v) with double deionized water. In a 500 ml glass media bottle, mix 368.4 ml of 95% ethanol with 131.6 ml of double deionized water.
D. Bleach Solution
0.5% Sodium hypochlorite (undiluted Clorox) in a 2 L Erlenmeyer flask.
E. 0.4% Trypan Blue Solution
Weigh out 0.2 g of trypan blue (Sigma, St. Louis, Mo.) and place it in a sterile 100 ml glass media bottle. Under sterile conditions using a 25 ml pipet, add 50 ml of Dulbecco's phosphate buffered saline (DPBS) with calcium chloride and magnesium chloride (GIBCO/Invitrogen Corporation, Carlsbad, Calif.) containing 1% of the preferred antibiotic-antimycotic solution (Mediatech [Cellgro], Herndon, Va.), at pH 7.4. Swirl the bottle gently to dissolve the trypan blue powder. Filter sterilize the trypan blue solution by passing it through a 0.2 μm bottle-top vacuum filter (VWR, Bristol, Conn.) into a 100 ml glass media bottle.
F. 8% Gelatin Solution
Weigh 4 g of gelatin granules (type-I collagen, Gelatin NF, EM Science, Gibbstown, N.J.). Add the gelatin granules to a sterile 100 ml media bottle. Next, under sterile conditions, add 50 ml of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Swirl the bottle gently to dissolve the contents of the bottle. Loosely tighten the cap of the bottle and place the bottle in the autoclave. Autoclave the mixture on the 15 minute liquid cycle. Once the autoclave cycle is complete, carefully remove the solution and allow it to cool to 40° C.
G. 8% Human Type-I Collagen Solution
Weigh 4 g of human type-I collagen. Add the collagen to a sterile 100 ml media bottle. Next, under sterile conditions, add 50 ml of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Swirl the bottle gently to dissolve the contents of the bottle. Loosely tighten the cap of the bottle and place the bottle in the autoclave. Autoclave the mixture on the 15 minute liquid cycle. Once the autoclave cycle is complete, carefully remove the solution and allow it to cool to 40° C.
H. 16% Gelatin Solution
Weigh 8 g of gelatin granules (type-I collagen, Gelatin NF, EM Science, Gibbstown, N.J.). Add the gelatin granules to a sterile 100 ml media bottle. Next, under sterile conditions, add 50 ml of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Swirl the bottle gently to dissolve the contents of the bottle. Loosely tighten the cap of the bottle and place the bottle in the autoclave. Autoclave the mixture on the 15 minute liquid cycle. Once the autoclave cycle is complete, carefully remove the solution and allow it to cool to 40° C.
I. 16% Human Type-I Collagen Solution
Weigh 8 g of human type-I collagen. Add the collagen to a sterile 100 ml media bottle. Next, under sterile conditions, add 50 ml of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Swirl the bottle gently to dissolve the contents of the bottle. Loosely tighten the cap of the bottle and place the bottle in the autoclave. Autoclave the mixture on the 15 minute liquid cycle. Once the autoclave cycle is complete, carefully remove the solution and allow it to cool to 40° C.
J. Sterile 5M Sodium Hydroxide
Weigh out 20 g of sodium hydroxide (Sigma, St. Louis, Mo.) granules and add them to a glass media bottle. Very slowly add 100 ml of double deionized water to the sodium hydroxide granules. Once the sodium hydroxide is dissolved, filter sterilize the solution by passing it through a 0.1 μm bottle top vacuum filter (VWR, Bristol, Conn.) into a 500 ml glass media bottle.
K. Sterile 5M Hydrochloric Acid
Measure 41.67 ml of double deionized distilled water. Add the water to a sterile 500 ml media bottle. Measure 208.33 ml of 6N hydrochloric acid (Sigma, St. Louis, Mo.). Very slowly add the hydrochloric acid to the bottle containing the water. Swirl the bottle gently to mix the contents. Filter sterilize the solution by passing it through a 0.1 μm bottle top vacuum filter (VWR, Bristol, Conn.) into a 500 ml glass media bottle.
L. PPELSC-DP-MACS Buffer
Under sterile conditions, take a fresh 500 ml bottle of Dulbecco's Phosphate Buffered Saline with calcium and magnesium (DPBS-Ca+2 & Mg+2) (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 0.5% w/v bovine serum albumin (Sigma, St. Louis, Mo.) and add 2 mM EDTA (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add a stirring bar to the mixture, and stir on a stirring plate at medium speed for 1 hour at ambient temperature. Filter the solution by passing it through a 0.45 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml bottle. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.2.
M. PPELSC-HC-MACS Buffer
Under sterile conditions, take a fresh 500 ml bottle of Dulbecco's Phosphate Buffered Saline with calcium and magnesium (DPBS-Ca+2 & Mg+2). Add 0.5% w/v human albumin and add 2 mM EDTA. Add a stirring bar to the mixture, and stir on a stirring plate at medium speed for 1 hour at ambient temperature. Filter the solution by passing it through a 0.45 μm bottle-top filter into a sterile 500 ml bottle. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.2.
N. 1.0 M Putrescine, Sterile
Weigh 1.61 g of putrescine (1,4-diaminobutane dihydrochloride) (Sigma, St. Louis, Mo.). Add the putrescine to a sterile 15 ml conical tube. Next, under sterile conditions, add 10 ml of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Invert tube to dissolve contents. Place contents in a 20 cc sterile syringe. Filter sterilize through a 0.2 μm syringe filter (VWR, Bristol, Conn.). Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
O. SFD-PPELSC-DP/HC-Tissue Wash
Under sterile conditions, take a fresh 500 ml bottle of DPBS-Ca+2 & Mg+2, (GIBCO/Invitrogen Corporation, Carlsbad, Calif.) discard 16 ml by placing it in the above-described bleach solution, and then add 15 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]), 0.1 ml of the above-described 1 M putrescine solution, and 0.9 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Swirl the bottle gently to mix the contents. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
P. Fibronectin Solution
Dissolve 5 mg of sterile fibronectin (Sigma, St. Louis, Mo.) in 5 ml of sterile OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Gently swirl bottle to dissolve.
Q. SFD-PPELSC-DP-Harvest and Storage Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Discard 15.6 ml by placing it in the above-described bleach solution, and then add 15 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 5 g of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Add 0.625 ml of the above-described 8% gelatin solution. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all the components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
R. SFD-PPELSC-HC-Harvest and Storage Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX. Discard 0.6 ml by placing it in the above-described bleach solution, and then add 0.1 ml of the above-described 1 M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Add 5 g of human serum albumin. Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Add 0.625 ml of the above-described 8% human type-I collagen solution. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all the components are dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
S. SFD-PPELSC-DP-Cell Isolation Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Discard 15.6 ml by placing it in the above-described bleach solution, and then add 15 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 100 μl of the above-described 1M putrescine (Sigma, St. Louis, Mo.) solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 5 g of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Add 0.625 ml of the above-described 8% gelatin solution. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all the components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4 forming an intermediate medium.
Weigh out 0.304 g, 75,000 units, of type-I collagenase (Worthington Biochemical Corporation, Lakewood, N.J.). Add the collagenase to a sterile 500 ml glass media bottle. Next, under sterile conditions, add 100 ml of the above-described intermediate medium. Add a sterile stirring bar to the mixture, and stir on a stirring plate at medium speed for 1 hour at ambient temperature. Filter the collagenase after one hour. Label four sterile 500 ml media bottles #1-4. Sterilize a BD Falcon bottle top-filter apparatus (VWR, Bristol, Conn.) with the above-described 70% ethanol solution. Once the ethanol has evaporated, assemble a BD Falcon bottle-top filter apparatus with a glass microfiber pre-filter and place it on bottle #1. Attach the tubing from a vacuum pump and vacuum filter the collagenase solution. Sterilize a BD Falcon bottle-top filter apparatus with the above-described 70% ethanol solution. Once the ethanol has evaporated, assemble a BD Falcon bottle-top filter apparatus with a glass microfiber pre-filter and place it on bottle #2. Attach the tubing from a vacuum pump and vacuum filter the collagenase solution a second time. Then, assemble another 70% ethanol sterilized BD Falcon bottle-top filter apparatus with a 0.45 μm filter (VWR, Bristol, Conn.) and a pre-filter and place it on bottle #3. Attach the tubing from a vacuum pump and filter the collagenase solution through the 0.45 μm filter. Filter the collagenase solution through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into bottle #4. The collagenase solution is now sterile and prepared for introduction into a dispase solution (Collaborative Biomedical Products, Bedford, Mass.). Add 100 ml of the dispase solution to another 500 ml sterile media bottle. Next, add 50 ml of sterile collagenase solution to the bottle containing 100 ml of dispase solution. This gives a final concentration of 250 units/ml of collagenase+33.3 units/ml of dispase. Aliquot 10 ml of the collagenase/dispase solution into 15×15 ml centrifuge tubes. Store the tubes at −20° C. until needed.
T. SFD-PPELSC-HC-Cell Isolation Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX. Discard 0.6 ml by placing it in the above-described bleach solution, and then add 100 μl of the above-described 1M putrescine solution. Add 0.5 ml of 55 mM 2-mercaptoethanol. Add 5 g of human albumin. Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Add 0.625 ml of the above-described 8% human type-I collagen solution. Add 37.5 μl of sterile fibronectin solution. Stir the solution on medium speed until all the components are dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4 forming an intermediate medium.
Weigh out 0.304 g, 75,000 units, of type-I collagenase. Add the collagenase to a sterile 500 ml glass media bottle. Next, under sterile conditions, add 100 ml of the above-described intermediate medium. Add a sterile stirring bar to the mixture, and stir on a stirring plate at medium speed for 1 hour at ambient temperature. Filter the collagenase after one hour. Label four sterile 500 ml media bottles #1-4. Sterilize a BD Falcon bottle top-filter apparatus with the above-described 70% ethanol solution. Once the ethanol has evaporated, assemble a BD Falcon bottle-top filter apparatus with a glass microfiber pre-filter and place it on bottle #1. Attach the tubing from a vacuum pump and vacuum filter the collagenase solution. Sterilize a BD Falcon bottle-top filter apparatus with the above-described 70% ethanol solution. Once the ethanol has evaporated, assemble a BD Falcon bottle-top filter apparatus with a glass microfiber pre-filter and place it on bottle #2. Attach the tubing from a vacuum pump and vacuum filter the collagenase solution a second time. Then, assemble another 70% ethanol sterilized BD Falcon bottle-top filter apparatus with a 0.45 μm filter and a pre-filter and place it on bottle #3. Attach the tubing from a vacuum pump and filter the collagenase solution through the 0.45 μm filter. Filter the collagenase solution through a 0.2 μm bottle-top filter into bottle #4. The collagenase solution is now sterile and prepared for introduction into a dispase solution, such as the above-described dispase solution. Add 100 ml of the dispase solution to another 500 ml sterile media bottle. Next, add 50 ml of sterile collagenase solution to the bottle containing 100 ml of dispase solution. This gives a final concentration of 250 units/ml of collagenase+33.3 units/ml of dispase. Aliquot 10 ml of the collagenase/dispase solution into 15×15 ml centrifuge tubes. Store the tubes at −20° C. until needed.
U. SFD-PPELSC-DP-Plating Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.), remove 31.25 ml, and then add 31.25 ml of the above-described 16% stock collagen solution. Swirl the bottle gently to mix the contents. Discard 5.6 ml by placing it in the above-described bleach solution, and then add 5 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 10 g of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all the components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4. Add 5 ng recombinant-human platelet-derived growth factor-BB (Collaborative Biomedical Products, Bedford, Mass.) per ml of solution. Swirl the bottle gently to mix the contents.
V. SFD-PPELSC-HC-Plating Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX, remove 31.25 ml, and then add 31.25 ml of the above-described 16% stock human type-I collagen solution. Swirl the bottle gently to mix the contents. Discard 0.6 ml by placing it in the above-described bleach solution, and then add 100 μl of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Add 10 g of human serum albumin. Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all the components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4. Add 5 ng recombinant-human platelet-derived growth factor-BB per ml of solution. Swirl the bottle gently to mix the contents.
W. SFD-PPELSC-DP-Propagation Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Discard 5.6 ml by placing it in the above-described bleach solution, and then add 5 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 1.0 g of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Add 0.625 ml of the above-described 8% gelatin solution. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all the components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4. Add 10.0 ng recombinant-human platelet-derived growth factor-BB (R&D Systems, Minneapolis Minn.) per ml of solution. Swirl the bottle gently to mix the contents.
X. SFD-PPELSC-HC-Propagation Medium
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX. Discard 0.6 ml by placing it in the above-described bleach solution, and then add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Add 1.0 g of human serum albumin. Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Add 0.625 ml of the above-described 8% human type-I collagen solution. Add 37.5 μl of the above-described sterile fibronectin solution. Stir the solution on medium speed until all components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4. Add 10.0 ng recombinant-human platelet-derived growth factor-BB per ml of solution. Swirl the bottle gently to mix the contents.
Y. SFD-PPELSC-DP-Cell Wash
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.), discard 5.6 ml by placing it in the above-described bleach solution, and then add 5 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 1% (w/v) of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
Z. SFD-PPELSC-HC-Cell Wash
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with L-glutamine, discard 0.6 ml by placing it in the above-described bleach solution, and then add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Add 1% (w/v) of human albumin. Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
AA. SFD-PPELSC-DP-Release Solution A
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.), discard 5.6 ml by placing it in the above-described bleach solution, and then add 5 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 10% (w/v) (50 g) of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
BB. SFD-PPELSC-HC-Release Solution A
Under sterile conditions, take a fresh 500 ml bottle of OPTI-MEM I with L-glutamine (Opti-MEM I with GLUTAMAX), discard 0.6 ml by placing it in the above-described bleach solution, and then add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Add 5% (w/v) (25 g) of human albumin. Add a sterile stirring bar. Stir the solution on medium speed until the albumin dissolves. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
CC. SFD-PPELSC-DP-Release Solution B
Under sterile conditions, take a fresh 500 ml bottle of Dulbecco's phosphate buffered saline (DPBS) with calcium and magnesium (GIBCO/Invitrogen Corporation, Carlsbad, Calif.), discard 5.6 ml by placing it in the above-described bleach solution, and then add 5 ml of the antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Invert the bottle a few times to mix the solution, and adjust the pH to 7.4 using the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid.
DD. SFD-PPELSC-HC-Release Solution B
Under sterile conditions, take a fresh 500 ml bottle of Dulbecco's phosphate buffered saline (DPBS) with calcium and magnesium, discard 0.6 ml by placing it in the above-described bleach solution, and then add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Invert the bottle a few times to mix the solution, and adjust the pH to 7.4 using the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid.
EE. SFD-PPELSC-DP-Release Solution C
Under sterile conditions, take a fresh 500 ml bottle of Dulbecco's phosphate buffered saline (DPBS) without calcium chloride and magnesium chloride (GIBCO/Invitrogen Corporation, Carlsbad, Calif.) and discard 7.8 ml by placing it in the above-described bleach solution. Next, add 5 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]) to the DPBS. Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Then add 2.2 ml of the above-described sterile 0.5M EDTA solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Invert the bottle a few times to mix the solution, and adjust the pH to 7.4 using the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid.
FF. SFD-PPELSC-HC-Release Solution C
Under sterile conditions, take a fresh 500 ml bottle of Dulbecco's phosphate buffered saline (DPBS) without calcium chloride and magnesium chloride and discard 2.8 ml by placing it in the above-described bleach solution. Add 0.1 ml of the above-described 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Then add 2.2 ml of the above-described sterile 0.5M EDTA solution. Invert the bottle a few times to mix the solution, and adjust the pH to 7.4 using the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid.
GG. SFD-PPELSC-DP-Release Solution D
Carefully weigh out 50 mg of trypsin (DIFCO, Becton-Dickinson Labware, Franklin Lakes, N.J.) and place it in a sterile 500 ml media bottle. Under sterile conditions, add 100 ml of the above-described SFD-PPELSC-DP-Release Solution C solution. Gently swirl the bottle to dissolve the trypsin. Once the trypsin has dissolved, filter sterilize the solution by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
HH. SFD-PPELSC-HC-Release Solution D
Carefully weigh out 50 mg of trypsin (DIFCO) and place it in a sterile 500 ml media bottle. Under sterile conditions, add 100 ml of the above-described SFD-PPELSC-HC-Release Solution C solution. Gently swirl the bottle to dissolve the trypsin. Once the trypsin has dissolved, filter sterilize the solution by passing it through a 0.1 μm bottle-top vacuum filter into a sterile 500 ml glass media bottle. Use the above-described sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4.
II. SFD-PPELSC-DP-Cryopreservation Medium
Under sterile conditions, take a fresh 500 ml bottle of Opti-MEM I with GLUTAMAX (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Discard 5.6 ml to bleach, and then add 5 ml of the preferred antibiotic-antimycotic solution (10,000 units/ml Penicillin G, 10,000 μg/ml Streptomycin, and 25 μg/ml Amphotericin B [Mediatech (Cellgro, Herndon, Va.); 100X]). Add 0.1 ml of 1M putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution (GIBCO/Invitrogen Corporation, Carlsbad, Calif.). Add 10 g of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.). Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Add 0.625 ml of the above-described 8% gelatin solution. Add 37.5 μg fibronectin (Sigma, St. Louis, Mo.). Stir the solution on medium speed until all the components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Filter sterilize the solution by passing it through a 0.1 μm bottle-top filter (VWR, Bristol, Conn.) into a sterile 500 ml glass media bottle. Use sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4. Remove 150 μl per ml medium and add 150 μl per ml 99.999% dimethylsulfoxide (Sigma, St. Louis, Mo.). Swirl gently to mix. Add 5 ng/ml recombinant-human platelet-derived growth factor-BB (Collaborative Biomedical Products, Bedford, Mass.). Swirl the bottle gently to mix the contents.
JJ. SFD-PPELSC-HC-Cryopreservation Medium
Under sterile conditions, take a fresh 500 ml bottle of Opti-MEM I with GLUTAMAX. Discard 0.6 ml to bleach, and then add 0.1 ml of the above-described 1M Putrescine solution. Add 0.5 ml of a 55 mM 2-mercaptoethanol solution. Add 10 g of human serum albumin. Add a sterile stirring bar. Stir the solution on medium speed until the BSA dissolves. Add 0.625 ml of the above-described 8% human type-I collagen solution. Add 37.5 μg fibronectin. Stir the solution on medium speed until all components have dissolved. Filter sterilize the solution by passing it through a 0.2 μm bottle-top filter into a sterile 500 ml glass media bottle. Filter sterilize the solution a second time by passing it through a 0.1 μm bottle-top filter into a sterile 500 ml glass media bottle. Use sterile 5M sodium hydroxide and/or sterile 5M hydrochloric acid to adjust the pH of the solution to 7.4. Remove 150 μl per ml medium and add 150 μl per ml 99.999% dimethylsulfoxide (Sigma, St. Louis, Mo.). Swirl gently to mix. Add 5 ng/ml recombinant-human platelet-derived growth factor-BB (Collaborative Biomedical Products, Bedford, Mass.). Swirl the bottle gently to mix the contents.
The present invention is described above and further illustrated below by way of examples, which are not to be construed in any way as imposing limitations upon the scope of the invention. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.
The following methods were used to isolate cells from animal tissue. Tissue harvested from a rat hind limb was used as a source for epiblast-like stem cells. (It should be noted that any organ or tissue with a connective tissue component can be utilized as a source for epiblast-like stem cells. However, the exact process for tissue removal will differ depending on the particular organ/tissue harvested.)
The following steps were taken:
The following method was used to isolate cells from human adult tissue. Tissue harvested from an adult human was used as a source for epiblast-like stem cells. (It should be noted that any organ or tissue with a connective tissue component can be utilized as a source for human adult stem cells. However, the exact process for tissue removal will differ depending on the particular organ/tissue harvested.)
The following steps were taken:
The following steps were taken to isolate stem cells using the animal and human tissue harvested in Examples 1 and 2 above.
Cell Isolation of Tissues Weighing 50 Grams or Less:
Pluripotent epiblast-like stem cells are not contact inhibited. Therefore, the cells continue to proliferate, forming multiple confluent layers of cells as long as the cells are maintained within proliferation medium.
Once multiple confluent layers of the cells are present, the cells are released from the surface of the flask using the following steps:
The pluripotent epiblast-like stem cells were cryopreserved by slow freezing and storage at −80° C. using the following protocol.
While the specification has been described in detail with respect to specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, the scope of the present invention should be assessed as that of the appended claims and any equivalents thereto.
This patent application claims the benefit of priority to U.S. provisional patent application Ser. No. 60/652,697 entitled “SERUM-FREE REAGENTS FOR THE ISOLATION, CULTIVATION, AND CRYOPRESERVATION OF POSTNATAL PLURIPOTENT EPIBLAST-LIKE STEM CELLS”, filed on Feb. 14, 2005, the subject matter of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60652697 | Feb 2005 | US |
