Method for Obtaining Xeno-Free Hbs Cell line
First Claim
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1. A method for obtaining a xeno-free hBS cell line, the method comprising the steps of:
- i) removing the zona pellucida from a blastocyst to obtain trophectoderm-enclosed inner cell mass by a xeno-free procedure,ii) at least partly removing the trophectoderm to obtain isolated inner cell mass cells by a xeno-free procedure,iii) placing the inner cell mass cells on a layer of human feeder cells in a xeno-free medium,iv) co-culturing of the inner cell mass cells with human feeder cells for a time period of from about 5 days to about 50 days in a xeno-free medium,v) releasing the inner cell mass cells or cells derived thereof from trophectoderm overgrowth, if any, by a xeno-free procedure,vi) selectively, transferring the inner cell mass cells or cells derived thereof to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells, andvii) propagating the xeno-free hBS cells by co-culturing with human feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line.
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Abstract
A method for obtaining a stable xeno-free hBS cell line, xeno-free hBS cell lines obtained according to said method and use thereof. The method comprises the steps of:
- i) removing the zona pellucida from a blastocyst to obtain trophectoderm-enclosed inner cell mass by a xeno-free procedure,
- ii) at least partly removing the trophectoderm to obtain isolated inner cell mass cells by a xeno-free procedure,
- iii) placing the inner cell mass cells on a layer of human feeder cells in a xeno-free medium,
- iv) co-culturing of the inner cell mass cells with human feeder cells for a time period of from about 5 days to about 50 days in a xeno-free medium,
- v) releasing the inner cell mass cells or cells derived thereof from trophectoderm overgrowth, if any, by a xeno-free procedure,
- vi) selectively, transferring the inner cell mass cells or cells derived thereof to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells,
- vii) propagating the xeno-free hBS cells by co-culturing with human feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line.
The xeno-free hBS cell line is suitable for use in medicine and in in vitro testing.
5 Citations
74 Claims
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1. A method for obtaining a xeno-free hBS cell line, the method comprising the steps of:
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i) removing the zona pellucida from a blastocyst to obtain trophectoderm-enclosed inner cell mass by a xeno-free procedure, ii) at least partly removing the trophectoderm to obtain isolated inner cell mass cells by a xeno-free procedure, iii) placing the inner cell mass cells on a layer of human feeder cells in a xeno-free medium, iv) co-culturing of the inner cell mass cells with human feeder cells for a time period of from about 5 days to about 50 days in a xeno-free medium, v) releasing the inner cell mass cells or cells derived thereof from trophectoderm overgrowth, if any, by a xeno-free procedure, vi) selectively, transferring the inner cell mass cells or cells derived thereof to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells, and vii) propagating the xeno-free hBS cells by co-culturing with human feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 61, 62, 63, 64, 65, 66, 68, 69, 71, 72, 73)
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31. A method according to step 30, wherein hBS cells are selectively passaged.
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59. A method for obtaining a xeno-free hBS cell line, the method comprising the steps of:
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1) removing the zona pellucida and at least a part of the trophectoderm from a blastocyst by incubation of the blastocyst with Acid Tyrodes Solution for a time period of from about 10 sec to about 10 min at room temperature to obtain isolated inner cell mass cells, 2) placing the inner cell mass cells on 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, β
-mercaptoethanol, penicillin and streptomycin,3) co-culturing of the inner cell mass cells with human foreskin fibroblast feeder cells in a xeno-free medium for a time period of from about 5 days to about 15 days changing at least 50% of the xeno-free medium every 3-5 days, 4) releasing the inner cell mass cells or cells derived thereof from trophectoderm overgrowth, if any, by using TrypLE™
Select (Invitrogen) as enzymatic treatment,5) selectively, transferring the inner cell mass cells or cells derived thereof to fresh layers of human foreskin fibroblast feeder cells in a xeno-free medium to obtain xeno-free hBS cells, 6) propagating the xeno-free hBS cells by co-culturing with human foreskin fibroblast feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line. - View Dependent Claims (60)
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67. (canceled)
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70. (canceled)
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74. (canceled)
Specification