WO2007042225A2 - A method for obtaining a xeno-free hbs cell line - Google Patents
A method for obtaining a xeno-free hbs cell line Download PDFInfo
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- WO2007042225A2 WO2007042225A2 PCT/EP2006/009697 EP2006009697W WO2007042225A2 WO 2007042225 A2 WO2007042225 A2 WO 2007042225A2 EP 2006009697 W EP2006009697 W EP 2006009697W WO 2007042225 A2 WO2007042225 A2 WO 2007042225A2
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- xeno
- cells
- free
- human
- recombinant
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1323—Adult fibroblasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Definitions
- the present invention relates to a method to obtain a stable xeno-free hBS cell line, xeno-free hBS cell lines obtained according to said method and use thereof.
- a stem cell is a cell type that has a unique capacity to renew itself and to give rise to specialized or differentiated cells. Although most cells of the body, such as heart cells or skin cells, are committed to conduct a specific function, a stem cell is uncommitted until it receives signals to develop into a specialized cell type. What makes the stem cells unique is their proliferative capacity, combined with their ability to become specialized. For years, researchers have focused on finding ways to use stem cells to replace cells and tissues that are lost, damaged or diseased. So far, most research has focused on two types of stem cells, embryonic and somatic stem cells. Embryonic stem cells are derived from the pre-implanted fertilized oocyte, i.e.
- blastocyst whereas the somatic stem cells are present in the adult organism, e.g. within the bone marrow, epidermis and intestine.
- Pluripotency tests have shown that whereas the embryonic or blastocyst-derived stem cells (hereafter referred to as blastocyst-derived stem cells or BS cells) can give rise to all cells in the organism, including the germ cells, somatic stem cells have a more limited repertoire in descendent cell types.
- hBS cells Perhaps the most far-reaching potential application of hBS cells is the generation of cells and tissue that could be used for so-called cell therapies. Many diseases and disorders result from disruption of cellular function or destruction of tissues of the body. Today, donated organs and tissues are often used to replace ailing or destroyed tissue. Unfortunately, the number of people suffering from disorders suitable for treatment by these methods by far outstrips the number of organs available for transplantation. The availability of hBS cells and the intense research on developing efficient methods for guiding these cells towards different cell fates, e.g. insulin-producing ⁇ -cells, cardiomyocytes, and dopamine-producing neurons, holds growing promise for future applications in cell-based treatment of degenerative diseases, such as diabetes, myocardial infarction and Parkinson's.
- degenerative diseases such as diabetes, myocardial infarction and Parkinson's.
- hBS human blastocyst-derived stem cell
- feeder-free matrices [Xu C et al, 2001], or feeder cells of human origin for derivation [Richards M et al, 2002, Inzunza J et al] and culture [Richards M et al, 2003] of hBS cell lines.
- three major hurdles have to be overcome.
- a protocol for the derivation of new hBS cell lines under xeno-free conditions must be developed.
- ICM inner cell mass
- xeno-free human feeder cells such as human foreskin fibroblast feeder cells have been reported before, although the feeders in many cases have been in contact with animal material, such as FBS during the actual establishment and culture of the feeder cells.
- the present invention relates to a method for obtaining a xeno-free hBS cell line, the method comprising the steps of:
- the present invention provides a method for obtaining a xeno- free hBS cell line as described above, but the starting point in the method may be any of the steps mentioned above, i.e. a method of the invention may comprise step ii)-step vii), step iii)-step vii), step iv)-step vii), step v)-step vii), step vi)-step vii) or step vii) (or step viii) as mentioned below).
- xeno-free is intended to mean never exposed to, directly or indirectly, material of non-human animal origin, such as cells, tissues, and/or body fluids and derivatives thereof.
- the method may further comprise a step
- the chosen time interval in step viii) is dependent on the cell growth.
- Step viii) mentioned above is also a specific aspect of the invention.
- the method according to the present invention may further comprise a step
- Step ix) may be performed in accordance with WO2004099394, which is hereby incorporated by reference.
- Step i) The zona pellucida is a thick extracellular matrix, rich in glycoprotein, surrounding the mammalian ovum (egg). Removal of the zona pellucida from a fertilized egg in order to obtain trophectoderm-enclosed inner cell mass in step i), can be performed by using a) an acidic solution, b) recombinant enzymes such as, e.g., hyaluronidase or pronase, or c) a mechanical procedure. The process of degradation of the zona pellucida can be followed by visual inspection in a microscope.
- trophectoderm-enclosed inner cell mass is intended to mean the material obtained after subjecting the fertilized egg to step I), wherein the zona pellucida is removed by either acidic hydrolysis, enzymatic digest or a mechanical procedure.
- the recombinant enzymes used herein have amino acid sequences which correspond to the human amino acid sequences for these enzymes, i.e. the recombinant enzymes used herein have human amino acid sequences but are produced recombinantly.
- the blastocyst is subjected to the acidic solution for about 5 seconds to about 180 seconds, such as, e.g., from about 10 seconds to about 120 seconds, from about 15 seconds to about 90 seconds, from about 20 seconds to about 60 seconds, from about 30 seconds to about 50 seconds.
- pH of the acidic solution is sufficiently low as to hydrolyse the carbohydrate structure of the zona pellucida, i.e. pH of the acidic solution is from about 2 to about 3, such as, e.g., from about 2.5 to about 2.8, such as 2.5. Any suitable acids can be used.
- the acidic solution is Acid Tyrodes solution with pH 2.5 +/- 0.3 (Sigma).
- the blastocyst is subjected to a solution of one or more recombinant enzymes. It is contemplated that the one or more recombinant enzymes have amino acid sequences which correspond to the human amino acid sequences for these enzymes.
- suitable enzymes are e.g. recombinant pronase, recombinant hyaluronidase and recombinant trypsin.
- suitable cell enzymatic solutions in step (i) are recombinant or xeno-free and potentially combining proteolytic and collagenolytic enzymes, such as e.g. AccutaseTM (Chemicon) and enzymatic solutions that are recombinant or xeno-free, potentially combining proteolytic, collagenolytic, and DNase activities, such as e.g. AccumaxTM (Innovative Cell Technologies).
- Suitable concentrations and treatment times are given in the following: Pronase: 10 U/ml, for a time period of from about 2 to about 20 minutes, such as from about 2 to about 10 min, from about 2 to about 5 min or from about 3-4 minutes (optimal).
- Hyaluronidase 70.000 U/ml, for a time period of from about 2 to about 240 minutes.
- Human recombinant trypsin 5-10.000 U, for a time period of from about 0.5 to about 30 minutes
- the zona pellucida can also be removed by a mechanical procedure, wherein for instance a glass capillary used under a visual inspection in the microscope.
- step ii) After removal of the zona pellucida, what is left of the blastocyst, i.e., trophectoderm- enclosed the inner cell mass, is subjected to step ii) in order to at least partly remove the trophectoderm.
- spontaneously hatched blastocysts can be subjected directly to step ii), thereby skipping step i).
- Step ii) can be performed by using a) an acidic solution, b) one or more recombinant enzymes, or c) a mechanical procedure.
- step ii) is performed by using an acidic solution
- the trophectoderm-enclosed inner cell mass obtained in step i) is subjected to the acidic solution for about 5 seconds to about 180 seconds, such as, e.g., from about 10 seconds to about 120 seconds, from about 15 seconds to about 90 seconds, from about 20 seconds to about 60 seconds, from about 30 seconds to about 50 seconds.
- pH of the acidic solution is sufficiently low as to hydrolyse the proteinaceous structure of the trophectoderm, i.e. pH of the acidic solution is from about 2 to about 3, such as from about 2.5 to about 2.8, such as 2.5. Any suitable acids can be used.
- the acidic solution is Acid Tyrodes solution with pH 2.5 +/- 0.3 (Sigma).
- the trophectoderm-enclosed inner cell mass is subjected to a solution of one or more recombinant enzymes. It is contemplated that the one or more recombinant enzymes are recombinant enzymes having an amino acid sequences corresponding to the human amino acid sequences for these enzymes.
- Suitable enzymes are recombinant proteolytic enzymes, such as, e.g., serine proteases including recombinant trypsin and TrypLETM Select.
- step ii) is typically performed by subjecting the trophectoderm-enclosed inner cell mass obtained in step i) to an undiluted ready-to-use concentration of TrypLETM Select for from about 0.5 to about 10 minutes, such as, e.g., from about 0.5 to about 8 minutes, from about 0.5 to about 5 minutes, from about 1 to about 3 minutes, such as for 1.5 minutes.
- step ii) is typically performed by subjecting the trophectoderm-enclosed inner cell mass obtained in step i) to from about 5.000 U to about 10.000 U of recombinant trypsin for from about 0.5 minutes to about 30 minutes.
- suitable cell enzymatic solutions in step (ii) are recombinant or xeno-free, potentially combining proteolytic and collagenolytic enzymes, such as e.g. AccutaseTM (Chemicon) and enzymatic solutions that are recombinant or xeno-free, potentially combining proteolytic, collagenolytic, and DNase activities, such as e.g. AccumaxTM (Innovative Cell Technologies).
- proteolytic and collagenolytic enzymes such as e.g. AccutaseTM (Chemicon)
- enzymatic solutions that are recombinant or xeno-free, potentially combining proteolytic, collagenolytic, and DNase activities such as e.g. AccumaxTM (Innovative Cell Technologies).
- the trophectoderm can also be at least partly removed by a mechanical procedure, which may be performed by using glass capillaries as a cutting tool.
- the detection of the inner cell mass cells is easily performed visually by microscopy.
- step iii) the resulting material after step ii) is placed on a human feeder layer typically by using a glass capillary under a microscope.
- the human feeder layer may be contained in suitable culture dishes such as, e.g., PRIMARIA® plastic dishes. If necessary the culture surface of the dishes may be coated with a suitable matrix material provided that this matrix material is xeno-free.
- a material suitable for use in this context includes recombinant human gelatin Step iv)
- the inner cell mass cells are co- cultured for a time period of from about 5 days to about 50 days, such as, e.g., from about 5 days to about 30 days, from about 5 days to about 20 days or from about 5 days to about 15 days in order to expand the cell population.
- the inner cell mass cells are co-cultured for a time period of 10 days in step iv).
- One or more medium changes may be performed during the co-cultivation of the inner cell mass cells in step iv) by changing from about 20% to about 100%, such as, e.g., from about 30% to about 80%, from about 40% to about 60% of the medium.
- the one or more medium changes are performed during the co-cultivation of the inner cell mass cells in step iv) by changing about 50% of the medium.
- the one or more medium changes may be performed at time intervals of from about 2 days to about 14 days, such as, e.g., from about 4 days to about 7 days.
- step iii) Since residual trophectodermal cells may have been placed on the layer of human feeder cells when placing the inner cell mass cells in step iii) visual inspection by microscopy can be performed at regular intervals in order to see whether the trophectoderm is interfering with the growth of the inner cell mass cells or inner cell mass derived cells.
- the suitable time point for taking the cells to step v) is when a considerable growth have been noticed over a couple of days by inspection in the microscope.
- the cell population obtained in step iv) may comprise inner cell mass cells as well as cells derived thereof, i.e. BS cells.
- the inner cell mass cells and cells derived thereof may be contaminated with residual trophectoderm. If that is the case, the trophectoderm tends to surround the inner cell mass cells or cells derived thereof.
- the inner cell mass cells or cells derived thereof can be released from such trophectoderm overgrowth by using a) a mechanical procedure or b) one or more recombinant enzymes.
- a suitable mechanical procedure is to use glass capillaries as a cutting tool.
- Inner cell mass cells or cells derived thereof are selectively cut out upon visual inspection in a microscope and transferred to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells (step vi).
- the inner cell mass cells or cells derived thereof may be released from trophectoderm overgrowth, if any, by using one or more recombinant enzymes, such as, e.g., one or more recombinant proteolytic enzymes including recombinant trypsin and T ⁇ ypLETM Select in step v). If recombinant trypsin is used for releasing the inner cell mass cells or cells derived thereof in step v), the inner cell mass cells or cells derived thereof are typically incubated with from about 5.000 U to about 10.000 U of recombinant trypsin for from about 0.5 minutes to about 30 minutes.
- recombinant enzymes such as, e.g., one or more recombinant proteolytic enzymes including recombinant trypsin and T ⁇ ypLETM Select in step v.
- the inner cell mass cells or cells derived thereof are typically incubated with from about 5.000 U to about 10.000 U of recombinant trypsin
- the inner cell mass cells or cells derived thereof are typically incubated with an undiluted ready-to-use concentration of TrypLETM Select for from about 0.5 to about 15 minutes.
- suitable cell enzymatic solutions in step (v) and (vi) are recombinant or xeno-free, potentially combining proteolytic and collagenolytic enzymes, such as e.g. AccutaseTM (Chemicon) and enzymatic solutions that are recombinant or xeno-free potentially combining proteolytic, collagenolytic, and DNase activities, such as e.g. AccumaxTM (Innovative Cell Technologies).
- proteolytic and collagenolytic enzymes such as e.g. AccutaseTM (Chemicon)
- enzymatic solutions that are recombinant or xeno-free potentially combining proteolytic, collagenolytic, and DNase activities such as e.g. AccumaxTM (Innovative Cell Technologies).
- the inner cell mass cells or cells derived thereof After having released the inner cell mass cells or cells derived thereof from trophectoderm, if any, the inner cell mass cells or cells derived thereof are selected upon visual inspection in a microscope and transferred to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells (step vi).
- step vii) the xeno-free hBS cells are propagated by co-culturing with feeder human cells to obtain a xeno-free hBS cell line.
- One or more passages can be performed during the propagation of the xeno-free hBS cells in step vii), wherein hBS cells are selectively passaged upon visual inspection in a microscope. These passages can be performed by using glass capillaries as a cutting tool.
- the one or more passages in step vii) can be performed by using one or more recombinant enzymes, such as, e.g., TrypLETM Select, recombinant trypsin, and/or recombinant collagenase.
- step vii) is typically performed by using an undiluted ready-to- use concentration of TrypLETM Select for from about 0.5 minute to about 15 minutes.
- step vii) is typically performed by using from about 5.000 U to about 10.000 U of recombinant trypsin for from about 0.5 minutes to about 30 minutes.
- step vii) is typically performed by using about 200 U/ml recombinant collagenase for from about 1 minute to about 40 minutes.
- suitable cell enzymatic solutions in step (vii) are recombinant or xeno-free potentially combining proteolytic and collagenolytic enzymes, such as e.g. AccutaseTM (Chmicon) and enzymatic solutions that are recombinant or xeno-free potentially combining proteolytic, collagenolytic, and DNase activities, such as e.g. AccumaxTM (Innovative Cell Technologies).
- the medium used in the individual passages may be the same or different.
- Propagation of the xeno-free hBS cell line is in accordance with the propagation described in step vii).
- passage of cells in step viii) may be performed by either mechanical dissection for example by using a glass capillary as cutting tool.
- the passage of cells in step viii) may be performed by using one or more recombinant enzymes, such as, e.g., TrypLETM Select, recombinant trypsin, and/or recombinant collagenase. Concentrations and incubation times for using TrypLETM Select, recombinant trypsin and recombinant collagenase, respectively, is as described for step vii).
- suitable cell enzymatic solutions in step (viii) are recombinant or xeno-free, potentially combining proteolytic and collagenolytic enzymes, such as e.g. AccutaseTM (Chemicon) and enzymatic solutions that are recombinant or xeno-free, potentially combining proteolytic, collagenolytic, and DNase activities, such as e.g. AccumaxTM (Innovative Cell Technologies).
- proteolytic and collagenolytic enzymes such as e.g. AccutaseTM (Chemicon)
- enzymatic solutions that are recombinant or xeno-free, potentially combining proteolytic, collagenolytic, and DNase activities such as e.g. AccumaxTM (Innovative Cell Technologies).
- the obtained hBS cell line is transferred to a xeno-free, feeder free culture system in step ix), said culture system comprising a suitable xeno-free support matrix such as, e.g., recombinant human gelatin, recombinant human fibronectin, human placental extracellular matrix and a suitable xeno-free medium which may be the same or different from the xeno-free medium employed upon establishment of the hBS cell line (steps iii), iv), vi), vii)) or upon maintenance on human feeder cells (step viii)).
- a suitable xeno-free support matrix such as, e.g., recombinant human gelatin, recombinant human fibronectin, human placental extracellular matrix and a suitable xeno-free medium which may be the same or different from the xeno-free medium employed upon establishment of the hBS cell line (steps iii), iv), vi), vii)) or
- the xeno-free medium can be conditioned by human feeder cells or it can be supplemented with suitable factors, such as, e.g., recombinant bFGF in high concentrations and/or activators of the WNT pathway.
- the human feeder cells used in any of steps iii), vi), vii) and viii) are obtained under xeno-free conditions.
- Human feeder cells are derived from healthy human tissue and can be obtained by biopsy.
- the human tissue from which the human feeder cells may be derived include embryonic, fetal, neonatal, juvenile or adult tissue, and it further includes tissue derived from skin, including foreskin, umbilical chord, muscle, lung, epithelium, placenta, fallopian tube, glandula, stroma or breast.
- the human feeder cells may be derived from cell types pertaining to the group consisting of human fibroblasts, fibrocytes, myocytes, keratinocytes, endothelial cells and epithelial cells.
- Examples of specific cell types that may be used for deriving human feeder cells include embryonic fibroblasts, extraembryonic endoderm cells, extraembryonic mesoderm cells, fetal fibroblasts and/or fibrocytes, fetal muscle cells, fetal skin cells, fetal lung cells, fetal endothelial cells, fetal epithelial cells, umbilical chord mesenchymal cells, placental fibroblasts and/or fibrocytes, placental endothelial cells, post-natal human foreskin fibroblasts and/or fibrocytes, post-natal muscle cells, post-natal skin cells, post-natal endothelial cells, adult skin fibroblasts and/or fibrocytes, adult muscle cells, adult fallopian tube endothelial cells, adult glandular endometrial cells, adult stromal endometrial cells, adult breast cancer parenchymal cells, adult endothelial cells, adult epithelial cells or adult keratinocytes.
- Feeder cells used in the present invention may further be immortalized or genetically modified. Immortalization of feeder cells means the acquisition of the ability to grow through an theoretically indefinite number of divisions in culture. There are several methods for doing that and one approach is to transforming the cells with e.g. viruses, retro viruses and/or by the expression of telomerase reverse transciptase protein (TERT). TERT is inactive in most cells, but when hTERT is exogenously expressed the cells are able to maintain telomere lengths sufficient to avoid replicative senescence.
- TERT telomerase reverse transciptase protein
- the feeder cells may be genetically modified having specific genes integrated to the genome. These genes may code for markers of interest or for synthesis of biomolecules known to be beneficial to the hBS cells, such as growth factors, such as e.g. bFGF (basic fibroblast growth factor).
- growth factors such as e.g. bFGF (basic fibroblast growth factor).
- the cells derived from hBS cells may be fibroblasts.
- the human feeder cells are derived from neonatal human foreskin.
- the human feeder cells are fibroblasts, preferably derived from human neonatal foreskin fibroblasts.
- Human foreskin samples can be obtained from circumcised baby boys. Samples may be aseptically selected in a sterile suitable medium, such as in sterile IMDM medium (Invitrogen) containing 2X Gentamycin (Invitrogen). Skin explants are placed in tissue culture flasks, such as, e.g., 25cm 2 primaria tissue culture flasks (Becton Dickinson), containing IMDM medium (Invitrogen), 1% penicillin-streptomyocin (Gibco Invitrogen Corporation) and 10% of human serum (Tallheden et al, 2005). After approximately 10 days, a confluent monolayer is established.
- tissue culture flasks such as, e.g., 25cm 2 primaria tissue culture flasks (Becton Dickinson)
- IMDM medium Invitrogen
- penicillin-streptomyocin Gibco Invitrogen Corporation
- human serum Teallheden e
- the cells were serially passaged using TrypLETM Select (Invitrogen). The inventors have found that a suitable time period between each passage is from about 2 to about 20 days and normally at least 15 passages are suitable such as maximal 20 passages. After expansion they were tested for a standard panel of human pathogens, including mycoplasma, HIV of type 1 and 2, Hepatitis of type B and C, Cytomegalovirus, Herpes Simplex Virus type 1 and 2, Epstein-Barr virus and Human Pailloma virus). Xeno-free medium
- the medium can be any base medium suitable for propagation of human inner cell mass cells.
- One suitable medium is Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1-30% v/v human serum and 2-100 ng/ml recombinant bFGF.
- the base medium comprises 20% v/v human serum.
- the base medium comprises 10 ng/ml recombinant bFGF.
- other base mediums can be used for as long as they provide the inner cell mass cells base with nutritional ingredients in a liquid form, i.e. inorganic ingredients such as trace elements and organic ingredients such as amino acids, salts, vitamins, energy providers, carbohydrates including sugars etc.
- the medium is xeno-free.
- the xeno-free medium used in any of the steps iii), iv), vi), vii) and/or viii) comprises a base medium suitable for propagation of human inner cell mass cells.
- a base medium is Dulbecco's Modified Eagle's Medium (DMEM).
- DMEM Dulbecco's Modified Eagle's Medium
- other base media may work as well.
- the xenofree medium may further comprise human serum, recombinant bFGF, L-glutamine or glutamax, nonessential amino acids, ⁇ -mercaptoethanol, penicillin and/or streptomycin.
- the concentration of human serum in the xeno-free medium is preferably from about 1% v/v to about 30% v/v human serum, such as, e.g., from about 10% v/v to about 30% v/v human serum, from about 15% v/v to about 25% v/v human serum, and more preferably 20% v/v human serum.
- the concentration of recombinant bFGF in the xeno-free medium is preferably from about 2 ng/ml to about 100 ng/ml recombinant bFGF, such as, e.g., from about 5 ng/ml to about 50 ng/ml recombinant bFGF, from about 5 ng/ml to about 25 ng/ml recombinant bFGF, from about 5 ng/ml to about 15 ng/ml recombinant bFGF, such as, e.g., 10 ng/ml recombinant bFGF.
- the concentration of L-glutamine or Glutamax® in the xeno-free medium is preferably from about 0.5 mM to about 20 mM, such as, e.g., from about 0.75 mM to about 10 mM, from about 1 mM to about 5 mM, such as, e.g. 2 mM.
- the concentration of non-essential amino acids in the xeno-free medium is preferably from about 0.01 mM to about 1 mM, such as, e.g., from about 0.03 noM to about 0.8 mM, from about 0.05 mM to about 0.6 mM, from about 0.07 mM to about 0.4 mM, from about 0.09 mM to about 0.2 mM, such as, e.g. 0.1 mM.
- the concentration of beta-mercaptoethanol in the xeno-free medium is preferably from about 10 ⁇ M to about 200 ⁇ M, such as, e.g., from about 25 ⁇ M to about 175 ⁇ M, from about 50 ⁇ M to about 150 ⁇ M, from about 75 ⁇ M to about 125 ⁇ M, such as, e.g., 100 ⁇ M.
- the concentration of penicillin in the xeno-free medium is preferably from about 5 units/ml to about 200 units/ml, such as, e.g., from about 10 units/ml to about 150 units/ml, from about 25 units/ml to about 100 units/ml, from about 25 units/ml to about 75 units/ml, such as, e.g., 50 units/ml.
- the concentration of streptomycin in the xeno-free medium is preferably from about 5 ⁇ g/ml to about 200 ⁇ g/ml, such as, e.g., from about 10 ⁇ g/ml to about 150 ⁇ g/ml, from about 25 ⁇ g/ml to about 100 ⁇ g/ml, from about 25 ⁇ g/ml to about 75 ⁇ g/ml, such as, e.g., 50 ⁇ g/ml.
- xeno-free media may be used for one or more individual steps of the present invention, such as in the propagation steps (vii) and (viii).
- Such medium may comprise salts, vitamins, an energy source (such as glucose) minerals, and amino acids.
- Suitable growth factors to be added to the medium could be e.g. GABA, pipecholic acid, lithium chloride, and transforming growth factor beta (TGF ⁇ ), and bFGF.
- the xeno-free hBS cell line derived in the present invention may be sub-cultured or propagated in media other than serum-comprising medium.
- the human serum used in any of steps iii), iv), vi), vii) and viii) is prepared by the following steps a) collecting healthy human blood in not-heparin coated bags, b) agitating the not-heparin coated bags for a time period of from about 0.5 hours to about 5 hours, such as, e.g., from about 0.5 hours to about 2 hours, c) incubating the not-heparin coated bags at a temperature of at the most 5° C for a time period of at least 10 hours d) optionally, selection based on clotting quality such as, e.g., absence of non-clotted fibrin, opacity of the liquid phase. e) separating the serum from the clotted material f) sterile filtrating the serum obtained in step d) g) pooling serum from at least 15 donors h) freezing serum before use.
- the method for obtaining a xeno-free hBS cell line comprises the steps of:
- a layer of human foreskin fibroblast feeder cells in a xeno-free medium comprising DMEM, human serum, recombinant bFGF, L-glutamine or glutamax, non-essential amino acids, ⁇ -mercaptoethanol, penicillin and streptomycin.
- the present invention provides a method for obtaining a xeno- free hBS cell line as described above, but the starting point in the method may be any of the steps mentioned above, i.e. a method of the invention may comprise step 2)-step 6), step 3)-step 6), step 4)-step 6), step 5)-step 6), or step 6) (or step 7) as mentioned below).
- Removal of the zona pellucida in step 1) can be followed by visual inspection in a microscope.
- step 3 visual inspection may be performed at regular intervals in order to see whether trophectoderm is interfering with the growth of the inner cell mass cells or cells derived thereof.
- the inner cell mass cells or cells derived thereof are selectively transferred in step 5) by using a glass capillary as cutting tool upon visual inspection in a microscope.
- the passage intervals suitable for the propagation of the xeno-free hBS cell line in step 7), depends on the cell growth and are performed by manual transfer using glass capillary.
- Another aspect of the present invention relates to a method for obtaining a xeno-free hBS cell line, comprising the steps of:
- Still another aspect of the present invention relates to a method for obtaining a xeno- free hBS cell line, comprising the steps of:
- Yet another aspect of the present invention relates to a method for obtaining a xeno- free hBS cell line, comprising the steps of:
- step vii) propagating the xeno-free hBS cells by co-culturing with feeder human cells in a xeno-free medium to obtain a xeno-free hBS cell line.
- the present invention also relates to propagation and maintenance culture of a xeno- free hBS cell line independently of the method for obtaining said xeno-free hBS cell line.
- the human serum based medium and human feeder cells as described herein may apply mutatis mutandis to the propagation method. More details appear from the description under step vii) and step viii).
- the present invention further relates to a xeno-free hBS cell line obtained by a method according to the invention.
- a xeno-free hBS cell line obtained by a method according to the invention.
- Such xeno-free hBS cell line has never been directly or indirectly exposed to any non-human animal material, meaning that all components in all steps of the method has not been exposed to any non-human animal material either, such as, e.g., any material derived from a non-human mammal.
- the human feeder cells used according to the present invention have been derived without any exposure to any non-human animal material.
- Any organic material used during establishment and maintenance of the xeno-free hBS cell line is of human origin or is synthetic, semi-synthetic or recombinant material.
- a xeno-free hBS cell line according to the present invention maintains self-renewal and pluripotency for a suitable period of time and, accordingly it is stable for a suitable period of time.
- the term "stable" is intended to denote proliferation capacity in an undifferentiated state for more than 50 weeks, such as, e.g., more than 40 weeks, more than 30 weeks, more than 20 weeks, more than 15 weeks, when grown on a layer of human feeder cells according to the invention.
- a xeno- free hBS cell line according to the invention is theoretically immortal.
- a xeno-free hBS cell line obtained by the method according to the method of the present invention can be used for the preparation of differentiated cells. Therefore the invention also relates to such differentiated cells.
- a xeno-free hBS cell or cell line according to the invention is capable of undergoing freezing and thawing.
- the xeno-free hBS cell line obtained in the present invention can be frozen and thawed according a vitrification method previously presented by Cellartis, WO2004098285, which is hereby incorporated by reference.
- the cells obtained in the present invention can be subject for clonal derivation as described in WO2005059116, which is hereby incorporated by reference.
- the xeno-free hBS cell line obtained by the present invention fulfils the general requirements.
- the cell line has one or more of the following characteristic, notably at least 4, 5, 6, 7 or 8 of the following characteristics.
- the cell line i) exhibits proliferation capacity in an undifferentiated state for more than 15 weeks when grown on mitotically inactivated feeder cells, and/or ii) exhibits normal euploid chromosomal karyotype, and/or iii) exhibits stable chromosomal karyotype during culture and/or iv) maintains potential to develop into derivatives of all types of germ layers both in vitro and in vivo, and/or v) exhibits at least two of the following molecular markers OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 , and the protein core of a keratin sulfate/chondroitin sulfate pericellular matrix
- a xeno-free hBS cell line according to the invention displays at least one, such as, e.g., at least two, at least three, at least four, at least five, or at least six of the following criteria; positive reactions for Oct-4, TRA-1-60, TRA-1-81 , SSEA-3 and SSEA-4 and negative reaction for SSEA-1.
- a xeno-free hBS cell lines according to the invention may be analysed for the immunohistochemical markers for undifferentiated cells, Oct-4, TRA-1-60, TRA-1-81 , SSEA-1 , SSEA-3 and SSEA-4 in order to monitor their state of differentiation.
- Alkaline phospatase and telomerase activity are often regarded as markers for undifferentiated hBS cells. Activity can be measured by any commercial kit available.
- the pluripotency of xeno-free hBS cell lines can be tested by letting the colonies differentiate spontaneously in the culture dishes for approximately 3-4 weeks of culture with medium changes every 2-7 days. The colonies are analysed by immunohistochemistry in order to identify cells from the three different germlayers. Suitable antibodies may be betatubulin for ectoderm, ASMA (alpha smooth muscle actin) for mesoderm and HNF3beta (hepatic nucleofactor 3 beta) for endoderm.
- One method to analyze if a human BS cell line has remained pluripotent is to xenograft the cells to immunodeficient mice in order to obtain tumors, teratomas.
- Various types of tissues found in the tumor should represent all three germlayers.
- Severe combined immunodeficient (SCID) -mice a strain that lack B- and T- lymphocytes can be used for analysis of teratoma formation.
- Human BS cells can be surgically placed in either testis or under the kidney capsule. In testis or kidney, BS cells can be transplanted in the range of 10 000-100 000 cells. Tumors are usually palpable after approximate 1 month. The mice are then sacrificed after 1-4 months and tumors dissected and fixed for either paraffin-or freeze-sectioning. The tumor tissue is subsequently analyzed by immunohistochemical methods. Specific markers for all three germlayers can be used.
- Chromosomes of the xeno-free hBS cell lines to be tested can be visualized using trypsin-Giemsa or DAPI staining.
- FISH fluorescence in situ hybridization
- kits containing probes for chromosomes 12, 13, 17, 18, 20, 21 and the sex chromosomes (X and Y) can be used according to the manufacturer's instruction with minor modifications. Slides can further be analyzed in an inverted microscope equipped with appropriate filters and software.
- Stem cells, and blastocyst-derived stem cells may further be characterized for their activity of the enzyme telomerase, which can be tested for with e.g. a kit called Telomerase PCR ELISA kit (Roche).
- the kit uses the internal activity of telomerase by amplification of the product by polymerase chain reaction (PCR) and detection of it with an enzyme linked immunosorbent assay (ELISA). Telomerase activity may as well be measured by QPCR.
- the differentiation status of the cells in the present invention can furthermore be tested by QPCR for specific genes.
- Undifferentiated or differentiated hBS cell colonies may be detached from the culture plate mechanically as whole colonies and washed in PBS and stored in -80 0 C.
- RNA may further be extracted using e.g. Qiagen RNeasy Mini Kit according to the manufacturer's instructions. Reverse transcription is performed using a suitable kit therefore, such as Bio-Rad iScript First Strand Synthesis Kit (according to the manufacturer's instructions) in a Rotorgene 3000 (Corbett Research) and the QPCR is performed under suitable conditions. All genes may be quantified in the same run and - if possible - differentiation status of several samples can be compared by calculating mathematical indices for the individual samples based on the genetic markers. (More detailed protocols are described in WO2006094798.)
- the individual components used in the herein presented invention may prior to use, as well as the xeno-free hBS cell line once established, be tested for human pathogens, such as e.g. Mycoplasma, Human Immunodeficiency Virus type 1 and 2, Hepatitis B and C, Cytomegalovirus, Herpes Simplex Virus type 1 and 2, Epstein-Barr Virus, and Human Papilloma Virus.
- human pathogens such as e.g. Mycoplasma, Human Immunodeficiency Virus type 1 and 2, Hepatitis B and C, Cytomegalovirus, Herpes Simplex Virus type 1 and 2, Epstein-Barr Virus, and Human Papilloma Virus.
- human pathogens such as e.g. Mycoplasma, Human Immunodeficiency Virus type 1 and 2, Hepatitis B and C, Cytomegalovirus, Herpes Simplex Virus type 1 and 2, Epstein-Barr Virus, and Human
- Xeno-free hBS cells derived in accordance with the herein presented invention may further be tested for Sialic acid Neu5Gc which is a membrane bound sugar molecule. A negative result of this test could be seen as an indication that no direct or indirect exposure to non-human animal material has occurred.
- a xeno-free hBS cell line according to the present invention can be used for the preparation of differentiated derivatives thereof, such as, e.g., progenitor cells of all three germ layers and more differentiated cells displaying characteristic features for different differentiated cell types, such as, e.g., hepatocyte-like features, cardiomyocyte-like features, neuron-like features.
- differentiated derivatives thereof such as, e.g., progenitor cells of all three germ layers and more differentiated cells displaying characteristic features for different differentiated cell types, such as, e.g., hepatocyte-like features, cardiomyocyte-like features, neuron-like features.
- a xeno-free hBS cell line according to the present invention may further be used for GMP production, such as clinical GMP production of hBS cells and/or differentiated cells thereof.
- GMP production such as clinical GMP production of hBS cells and/or differentiated cells thereof.
- the method for xeno-free derivation as described herein may be performed under GMP and/or cGMP conditions to provide clinically applicable cell lines and derivatives (Martin et al Nat. Med 2005).
- a xeno-free hBS cell line according to the invention, or differentiated derivatives thereof, can be used in medicine.
- a xeno-free hBS cell line, or differentiated derivatives thereof may be used for the manufacture of a medicinal product for the prevention and/or treatment of pathologies and/or diseases caused by tissue degeneration.
- a xeno-free hBS cell line, or differentiated derivatives thereof may be used for the manufacture of a medicinal product for the treatment and/or prevention of metabolic pathologies and/or diseases.
- the hBS cells obtained by a method according to the invention may be used for the manufacture of a medicament for transplantation of xeno-free hBS cells into a mammal for the prevention or treatment of a disease.
- a specific aspect is the use of xeno-free hBS cells in autologous transplantation, i.e. only human material from the specific patient in question has been used in the preparation of the xeno-freee hBS cells or cell line.
- xeno-free cells or cell lines according to the invention can be suitable for use including liver diseases, cardiovascular diseases including myocardial infarction; degenenerative diseases such as, e.g., neurodegenerative diseases including Parkinson's disease and Alzheimer's disease; diabetes.
- Such a medicament may comprise undifferentiated xeno-free hBS cells or differentiated xeno-free hBS cells dispersed in a pharmaceutically acceptable medium such as an aqueous medium.
- the medium may comprise one or more additive selected from the group consisting of pH adjusting agents, stabilizers, preservatives, osmotic pressure adjusting agent, and physiologically acceptable salts; and/or one or more agents selected from the group consisting of therapeutically active substances, prophylactically active substances, engraftment improving agents, viability improving agents, differentiation improving agent and immunosuppressive agents.
- WO2006034873 treatment of hepatic related diseases can be envisioned or after differentiating the cells towards a neural progenitor treatment of neural diseases, such as multiple sclerosis, hypoxic injury, ischemic injury, traumatic injury, Parkinson's disease, and demyelition disorder, can be envisioned.
- neural diseases such as multiple sclerosis, hypoxic injury, ischemic injury, traumatic injury, Parkinson's disease, and demyelition disorder
- Additional progenitor cell types derived from a xeno-free hBS cell line obtained as described herein may be mesodermal, with the potential to give rise to other than cardiac cell types also e.g. bone and cartilage, or endodermal with the potential to give rise to also pancreatic cell types, such as beta cells.
- hBS cell lines derived according to the method presented herein, it will be possible to use these cells for the derivation of progenitor cells that later can be transplanted and evaluated for the potential use in humans.
- progenitor cells could have the potential to in situ differentiate further into the cell type of the degenerated tissue, such as the site of a cardiac infarction.
- Cells differentiated from a xeno-free hBS cell line may in additon be used for treating disorders associated with, for example, necrotic, apoptotic, damaged, dysfunctional or morphologically abnormal myocardium.
- disorders include, but are not limited to, ischemic heart disease, cardiac infarction, rheumatic heart disease, endocarditis, autoimmune cardiac disease, valvular heart disease, congenital heart disorders, cardiac rhythm disorders, and cardiac insufficiency.
- cardiomyocytes including cardiomyocytes, endothelial cells and smooth muscle cells
- smooth muscle cells may therefore be suitable for treating the majority of cardiac disorders and diseases by reversing, inhibiting or preventing cardiac damage caused by ischemia resulting from myocardial infarction.
- the treatment is preferably performed by administering a therapeutically effective dose of the cells to the heart of the subject, preferably by injection into the heart.
- a therapeutically effective dose is an amount sufficient to generate a beneficial or desired clinical result, which dose could be administered in one or more administrations.
- the injection can be administered into various regions of the heart, depending on the type of cardiac tissue repair required.
- the administration may be performed using a catheter-based approach after opening up the chest cavity or entry through any suitable blood vessel.
- the effective dose of cells can be based on factors such as weight, age, physiological status, medical history, infarct size and elapsed time following onset of ischemia.
- the administration of the cells preferably comprises treating the subject with an immunosuppressive regimen, preferably prior to such administration, so as to inhibit such rejection.
- a xeno-free hBS cell line obtained by a method according to the present invention, or differentiated derivatives thereof, may also be used for medical research as they are very suitable for use in in vitro models for studying human diseases, such as, e.g., human degenerative diseases.
- Potential applications of xeno-free hBS cells themselves and cell lines and cell populations derived therefrom are found e. g. in the drug discovery and drug development processes in the pharmaceutical industry and in toxicity testings of all kinds of chemicals.
- Today, large-scale and high throughput screening of drug candidates usually relies on biochemical assays that provide information on compound binding affinity and specificity, but little or no information on function.
- Functional screening relies upon cell-based screens and usually uses organisms of poor clinical relevance such as bacteria or yeasts that can be produced cheaply and quickly at high volume. Successive rounds of screening use model species of greater clinical relevance, but these are more costly and the screening process is time consuming. Screening tools based on human primary cells or immortalised cell types exist, but these cells are limited in supply or usefulness due to loss of vital functions as a result of in vitro culture and transformation. The access to xeno-free hBS cells and hBS cells differentiated under engineered conditions provides a new and unique capability to conduct human cell-based assays with high capacity, but without compromising clinical relevance.
- Xeno-free hBS cells can be used in high throughput screenings by combining high capacity with improved clinical significance.
- the ability to precisely modify the genome using gene targeting in hBS cells with or without differentiation of the genetically modified cells into various cell types allows the application of this technology to the identification of novel therapeutically active substances through primary and secondary screening.
- the invention relates to the user of the hBS cells obtained by a method according to the invention defined for i) the production of monoclonal antibodies, ii) in vitro toxicity screening, iii) in vitro screening of potential drug substances, or iv) identification of potential drug substances.
- Figure 1 shows a blastocyst prior to treatment with acid Tyrode's solution to remove the zona pellucida and parts of the trophectoderm in 40 times magnification.
- Figure 2 shows a blastocyst after treatment with acid Tyrode's solution to remove the zona pellucida and parts of the trophectoderm in 40 times magnification.
- Figure 3 shows hBS cell line SA611 in A) passage 7, day 4 in 10 times magnification.
- Figure 4 shows positive reactions for the undifferentiated hBS cell markers A) ALP.
- Figure 5 shows positive reaction for the undifferentiated hBS cell marker SSEA-4 and negative reaction for the differentiated hBS cell marker SSEA-1 , both in passage 6 and in 20 times magnification.
- Figure 6 shows positive reactions for undifferentiated hBS cell markers Tra 1-60 and Tra 1-81 , both in passage 6 and in 20 times magnification.
- Figure 7 shows positive reactions for the endodermal marker HNF-3beta and the neuroectodermal marker beta-tubulin in passage 6 after undergoing spontaneous differentiation for 21-28 days, in 20 times magnification.
- Figure 8 shows additional morphological and immuhistochemical characterization of the xeno-free hBS cell line SA611.
- A shows the blastocyst (scale-bar 25 urn).
- C-H shows immunofluorescence of undifferentiated SA611 after 12 passages using Oct-4 (C), SSEA-1 (D), Tra1-60 (E), Tra1-81 (F), SSEA-3 (G), SSEA-4 (H).
- Scale bar 50 urn in (C), (E) and (G) and 100 urn in (F) and (H).
- Figure 9 shows genetic characterization of the xeno-free hBS cell line SA611 in passage 9.
- A shows that the chromosomes were diploid and normal.
- B shows fluorescence in situ hybridization of selected chromosomes from SA611 which demonstrates that the cells were XY and diploid for normal chromosomes 12 and 17.
- C further shows the X chromosome in blue, the Y chromosome in gold, chromosome 13 in red, chromosome 18 in aqua and chromosome 21 n green (which is nearly impossible to visualize in black and white).
- Figure 10 shows confirmation of pluripotency of the xeno-free hBSC line SA611 in vivo in (A), (C) and (E) and in vitro in (B), (D) and (F): Histological analysis of teratomas from SA611 after 11 passages under xeno-free conditions: (A) neuroectoderm (ectoderm), (C) cartilage (mesoderm), (E) secretory epithelium (endoderm).
- Example 1 Obtaining and culture xeno-free hBS cells
- the blastocyst was treated with acid Tyrode's solution (Medicult) solution (ready-to-use concentration) for 15-30 seconds in room temperature in order to remove the zona pellucida and parts of the trophectoderm (see figure 1 and 2) before placing the inner cell mass cells onto mitomycin-C inactivated xenofree human foreskin fibroblast feeders in xeno-free serum containing a DMEM with osmolarity of around 270, supplemented with 20% (v/v) human serum, 4 ng/mL human recombinant bFGF, 1% penicillin-streptomyocin, 1% Glutamax, 0.5 mmol/l ⁇ - mercaptoethanol and 1% non-essential amino acids (Gibco Invitrogen Corporation).
- Example 2 Establishment of a human foreskin fibroblast feeder cell line (such as cell line hFF003) Human foreskin samples were aseptically collected in sterile IMDM (Invitrogen) containing 2X Gentamycin from a circumcised 8 week old boy. Skin explants were placed inside 25cm2 primaria tissue culture flasks (Becton Dickinson) containing IMDM medium (Invitrogen), 1% penicillin-streptomyocin (Gibco Invitrogen Corporation) and 10% of human serum. After approximately 10 days, a confluent monolayer was established. The cells were serially passaged using TrypLETM Select (Invitrogen).
- tissue cultured wells Prior to plating the xenofree human fibroblast feeders, the tissue cultured wells are coated with 0.1% recombinant human gelatin (Fibrogen) for a minimum of 1 hour at room temperature. Confluent monolayers of xenofree hFF003 (fifth to eight passage) cells grown in IMDM, 10 % human serum and 1% penicillin-streptomyocin were then treated with mitomycin-C (Sigma) for 2.5 hours.
- Fibrogen recombinant human gelatin
- Mitomycin-C treated feeders were plated on IVF wells (Becton Dickinson), 200 000 cells per 2.89 cm2 in a medium which was based on DMEM (as above) supplemented with 10% (v/v) human serum, 1% penicillin-streptomyocin, 1% Glutamax, 0.5 mmol/l ⁇ -mercaptoethanol and 1% non- essential amino acids (Gibco Invitrogen Corporation).
- the medium Prior to the placing blastocysts with their inner cell mass cells and cells derived therefrom or hBS cells, the medium was changed to a DMEM (as above), now instead supplemented with 20% (v/v) human serum, 10 ng/mL human recombinant bFGF, 1% penicillin-streptomyocin, 1% Glutamax, 0.5 mmol/l ⁇ -mercaptoethanol and 1% non-essential amino acids (Gibco Invitrogen Corporation). (Same medium as described in Example 1).
- Human serum was obtained from blood-samples from at least 15 healthy individuals (Blodcentralen, Sahlgrenska University Hospital) by collecting the blood in non-heparin coated plastic bags at approximately 8 °C over night whereby the serum could be separated from the clotted material. The serum was further sterile filtered, pooled and frozen in suitable portions. (The blood prior to use was at Blodcentralen, Sahlgrenska University JHospital tested for the standard battery of pathogens including Hepatitis B, C, HIV, HTLV and syphilis.) The medium was further prepared by adding 20% (v/v) of the thawed serum to a DMEM (as above) together with the other ingredients as described in Example 1. Example 5 - Freezing and thawing by vitrification of xeno-free hBS cells
- the hBS cell line SA611 have been frozen and thawed in several passages, e.g. in passage 25 according to a method described in WO2004098285.
- Two solutions A and B are prepared (Solution A: Sterile filtered 10% Ethylene glycol, 10% DMSO in Cryo- PBS; Solution B: Sterile filtered 0.3M Trehalose, 20% Ethylene glycol, 10% DMSO in Cryo-PBS) Selected colonies of hBS cell line SA611 were cut in the same way as when the cells are cut for regular passage using a stem cell cutting tool (Swemed Labs International, Billdal, Sweden).
- the cell pieces are incubated first in 500 ml preheated (37°C) Solution A for 1 min and then transferred to 25 ml Solution B and incubated for 30 s and then transferred again to a fresh drop of Solution B and incubated for between 20 and 30 s.
- the volume was about 40-50 ⁇ l.
- the cell pieces were aspirated into a straw prepared for vitrification and the straw was then closed with a bond. The straw was further plunged into liquid nitrogen.
- the hBS cells were incubated for 1 min in 500 ⁇ l solution C and the transferred to 500 ⁇ l solution D and incubated for 5 min. Under a steromicroscope the hBS cell pieces were quickly rinsed in the xeno-free serum based medium and then seeded in a culture dish on top of xeno-free human fibroblast feeder cells in the serum based medium. The cells were then cultured (incubated at 37 0 C) and the number of established new colonies were counted and passaged in order to verify the viability of the hBS cells after vitrification.
- Example 6 Characterization of a xeno-free hBS cell line lmmunohistochemical and histochemical analysis
- the hBS cell colony cultures were fixed in 4% paraformaldehyde and subsequently permabilized. After consecutive washing and blocking steps, the cells were incubated with the primary antibody overnight at 4 0 C.
- the primary antibodies used were specific for Oct-4, TRA-1-60, TRA-1-81 , SSEA-1 , SSEA-3 and SSEA-4 (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.southernbiotech.com).
- FITC- or Cy3- conjugated secondary antibodies were used for detection. Nuclei were counterstained with DAPI (Vectashield; Vector Laboratories, Burlingame, CA; http://www.vectorlabs.com).
- the activity of alkaline phosphatese (ALP) was determined according to manufacturer's protocol (Sigma-Aldrich Sweden; http://www.sigmaaldrich.com).
- Alkaline phosphatase (ALP) reactions were detected using a commercial kit and following the manufacturer's protocol (Sigma-Aldrich). Positive reactions in undifferentiated colonies were detected for ALP (see figure 4) , Oct-4, Tra1-60, Tra 1-81 , SSEA-3, and SSEA-4 while SSEA-1 (see figures 5, 6, 8) was negative. (See figures 4-6.)
- Karyotyping and FISH hBS cells designated for genetic characterization were retransferred to mouse embryonic fibroblasts for two passages or transferred to Matrigel plates (Becton Dickinson) and further cultured for approximately 10 days. The cells were then incubated in the presence of Calyculin A, collected by centrifugation, lysed by hypotonic treatment, and fixed using ethanol and glacial acetic acid. The chromosomes were visualized using trypsin-Giemsa or DAPI staining.
- FISH fluorescence in situ hybridization
- the pluripotency of SA611 was initially tested in vitro by either allowing the hBS cell colonies to spontaneously differentiate on the feeders or by transfer the undifferentiated hBS cell colonies to MatrigelTM coated plates (Becton Dickinson) on which they were allowed to spontaneously differentiate. In both cases the medium was switched to VitroHESTM (Vitrolife, Kungsbacka, Sweden) when differentiation was to be induced. After 3-4 weeks of culture, the colonies were analysed by immunohistochemistry in order to identify cells from the three different germlayers.
- the xeno-free hBS cell line SA611 stably expressed the genetic and phenotypic characteristics of undifferentiated pluripotent human BS cells.
- WO2003055992 A method for the establishment of a pluripotent human blastocyst- derived stem cell line
- WO2006094798 Use of panel of pairs of primers complementary to reporter genes of cell differentiation Improved methods for the generation of hepatocyte-like cells from human blastocyst- derived stem (hBS) cells
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CA002624728A CA2624728A1 (en) | 2005-10-07 | 2006-10-06 | A method for obtaining a xeno-free hbs cell line |
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US12/083,192 US20100129906A1 (en) | 2005-10-07 | 2006-10-06 | Method for Obtaining Xeno-Free Hbs Cell line |
GB0807891A GB2445338A (en) | 2005-10-07 | 2006-10-06 | A method for obtaining a xeno-free HBS cell line |
EP06806097A EP1945758A2 (en) | 2005-10-07 | 2006-10-06 | A method for obtaining a xeno-free hbs cell line |
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WO2010149597A2 (en) | 2009-06-18 | 2010-12-29 | Cellartis Ab | 3D CULTURING SYSTEMS FOR GROWTH AND DIFFERENTIATION OF HUMAN PLURIPOTENT STEM (hPS) CELLS |
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US20080182328A1 (en) * | 2006-12-19 | 2008-07-31 | The Burnham Institute | Mammalian extraembryonic endoderm cells and methods of isolation |
JP5409222B2 (en) * | 2009-09-10 | 2014-02-05 | 学校法人慶應義塾 | Hematopoietic stem cell culture method |
US20160355781A1 (en) * | 2015-06-03 | 2016-12-08 | Texas Health Biomedical Advancement Center, Inc. | Systems, methods, and cellular compositions thereof, involving introduction, attachment and proliferation of trophectoderm cells in a blastocyst |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006029198A2 (en) * | 2004-09-08 | 2006-03-16 | Wisconsin Alumni Research Foundation | Culturing human embryonic stem cells |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6667176B1 (en) * | 2000-01-11 | 2003-12-23 | Geron Corporation | cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells |
US7005252B1 (en) * | 2000-03-09 | 2006-02-28 | Wisconsin Alumni Research Foundation | Serum free cultivation of primate embryonic stem cells |
IL160351A0 (en) * | 2001-08-23 | 2004-07-25 | Reliance Life Sciences Pvt Ltd | ISOLATION OF INNER CELL MASS FOR THE ESTABLISHMENT OF HUMAN EMBRYONIC STEM CELL (hESC) LINES |
US20050233446A1 (en) * | 2003-12-31 | 2005-10-20 | Parsons Xuejun H | Defined media for stem cell culture |
KR101264940B1 (en) * | 2004-09-08 | 2013-05-15 | 위스콘신 얼럼나이 리서어치 화운데이션 | Medium and culture of embryonic stem cells |
-
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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Non-Patent Citations (10)
Title |
---|
AMIT M ET AL: "Feeder layer- and serum-free culture of human embryonic stem cells" BIOLOGY OF REPRODUCTION, vol. 70, no. 3, March 2004 (2004-03), pages 837-845, XP002978624 ISSN: 0006-3363 * |
ELLERSTRÖM C ET AL: "Derivation of a xeno-free human embryonic stem cell line." STEM CELLS, vol. 24, no. 10, October 2006 (2006-10), pages 2170-2176, XP008072571 ISSN: 1066-5099 * |
INZUNZA J ET AL: "Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells." STEM CELLS, vol. 23, no. 4, April 2005 (2005-04), pages 544-549, XP002389652 ISSN: 1066-5099 cited in the application * |
KLIMANSKAYA I ET AL: "Human embryonic stem cells derived without feeder cells" THE LANCET, vol. 365, no. 9471, May 2005 (2005-05), pages 1636-1641, XP004882308 ISSN: 0140-6736 * |
LEE J B ET AL: "Available human feeder cells for the maintenance of human embryonic stem cells." REPRODUCTION, vol. 128, no. 6, December 2004 (2004-12), pages 727-735, XP002377143 ISSN: 1470-1626 * |
LUDWIG T E ET AL: "Derivation of human embryonic stem cells in defined conditions." NATURE BIOTECHNOLOGY, vol. 24, no. 2, February 2006 (2006-02), pages 185-187, XP002422483 ISSN: 1087-0156 * |
MALLON B S ET AL: "Toward xeno-free culture of human embryonic stem cells." THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, vol. 38, no. 7, 2006, pages 1063-1075, XP005362917 ISSN: 1357-2725 * |
MARTIN M J ET AL: "Human embryonic stem cells express an immunogenic nonhuman sialic acid." NATURE MEDICINE, vol. 11, no. 2, February 2005 (2005-02), pages 228-232, XP002389653 ISSN: 1078-8956 cited in the application * |
MOORE H: "The medium is the message." NATURE BIOTECHNOLOGY, vol. 24, no. 2, February 2006 (2006-02), pages 160-161, XP002422484 ISSN: 1087-0156 * |
RICHARDS M ET AL: "Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells." NATURE BIOTECHNOLOGY, vol. 20, no. 9, September 2002 (2002-09), pages 933-936, XP002335247 ISSN: 1087-0156 cited in the application * |
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GB0807891D0 (en) | 2008-06-04 |
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