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Culture system and method for propagation of human blastocyst-derived stem cells

A culture system and cell technology, applied in the field of culture systems for proliferating human blastocyst-derived stem cells, can solve the problems of a lot of man-hours, laboratory space, low compatibility, reproducibility, and negative effects of standardization

Inactive Publication Date: 2012-07-04
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This traditional culture method is time-consuming and laborious, but is by far the preferred culture method for maintaining hBS cell lines in a stable, normal state for long periods of time, and is therefore an appropriate method for smaller-scale maintenance cultures
However, there are also a number of technical drawbacks associated with this traditional culture system
For example, it is nearly impossible to quantify how many cells are transferred into a small plate, thus negatively affecting reproducibility and standardization
Scale-up production processes based on these traditional culture systems are limited due to low compatibility with automation technologies such as automated instruments and bioreactors, and instead require extensive man-hours, laboratory space and equipment such as microscopes

Method used

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  • Culture system and method for propagation of human blastocyst-derived stem cells
  • Culture system and method for propagation of human blastocyst-derived stem cells
  • Culture system and method for propagation of human blastocyst-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Culture of human foreskin fibroblast feeder cells and use as feeder cell layer

[0152] Commercially available hFFs were obtained from the American Type Culture Collection (CRL-2429 ATCC, Manassas, VA) and cultured in a medium supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin. Iscove's DMEM (Gibco Invitrogen Corporation, Paisley, Scotland; http: / / www.invitrogen.com). Cells were passaged periodically (weekly) using trypsin-EDTA (Invitrogen) at a split ratio between 1:2 and 1:8. Confluent monolayers of hFF were treated with mitomycin-C (Sigma) (10 μg / ml, 2.5 hours) and incubated in VitroHES supplemented with 4 ng / ml human recombinant basic fibroblast growth factor (hrbFGF, Invitrogen). TM 70,000-80,000 cells / cm in medium 2 at a density of 0.1% gelatin (Sigma) coated IVF dishes. hFF cells were used as feeder cells between passage 4 and passage 12.

Embodiment 2

[0154] Culture of human embryonic fibroblasts and use as feeder cell layer

[0155] Commercially available human embryonic fibroblasts were obtained from the American Type Culture Collection (CCL-110 ATCC, Manassas, VA) and cultured in Iscove's supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin. DMEM (Gibco Invitrogen Corporation, Paisley, Scotland; http: / / www.invitrogen.com). Cells were passaged periodically (weekly) using trypsin-EDTA (Invitrogen) at a split ratio between 1:2 and 1:8. Confluent monolayers of human embryonic fibroblasts were treated with mitomycin-C (Sigma) (10 μg / ml, 2.5 hours) and treated with 4 ng / ml human recombinant basic fibroblast growth factor (hrbFGF, Invitrogen ) VitroHES TM 70,000-80,000 cells / cm in medium 2 at a density of 0.1% gelatin (Sigma) coated IVF dishes. Human embryonic fibroblasts were used as feeder cells between passage 4 and passage 12, Figure 8 .

Embodiment 3

[0157] Establishment of human foreskin fibroblast feeder cell lines (e.g. cell line hFF003)

[0158]Human foreskin samples from circumcised 8-week-old boys were aseptically collected in sterile IMDM (Invitrogen) containing 2X gentamicin. Skin explants were placed in 25 cm2 primary tissue culture flasks (Becton Dickinson) containing IMDM medium (Invitrogen), 1% penicillin-streptomycin (Gibco Invitrogen Corporation) and 10% human serum. After about 10 days, a confluent monolayer was established. Use TrypLE TM Cells were serially passaged with Select (Invitrogen). After expansion, for a standard panel of human pathogens (mycoplasma, HIV types 1 and 2, hepatitis B and C, cytomegalovirus, herpes simplex virus types 1 and 2, Epstein-Barr virus, human papillomavirus) They were tested and all came back negative.

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Abstract

The present invention relates to a culture system for and a method for propagation of human blastocyst-derived stem cells (hBS cells) upon enzymatic dissociation into a single cell suspension. The culture system for propagation of human blastocyst-derived stem (hBS) cells comprises i) human feeder cells at a density of at least 50,000 cells / cm<2>, ii) one or more dissociation agents for dissociation of hBS cell colonies into a single cell suspension, and iii) a supportive culture medium, which culture system makes it possible to propagate hBS cells by dissociation of hBS cell colonies into a single cell suspension at each consecutive passage for an extended time period, while maintaining the significant characteristics of hBS cells.

Description

field of invention [0001] The present invention relates to culture systems and methods for the proliferation of human blastocyst-derived stem cells (hBS cells) based on enzymatic dissociation into single cell suspensions. Background of the invention [0002] Human blastocyst-derived stem cells (hBS cells) are traditionally maintained on mEF (mouse embryonic fibroblast) feeder cell layers and propagated by manual dissection and transfer of individual pieces of colonies [Heins et al., WO03055992, Bresagen ]. This traditional culture method is very time-consuming and labor-intensive, but it is by far the preferred culture method for maintaining hBS cell lines in a stable normal state for a long period of time, and is therefore suitable for smaller scale maintenance cultures. However, there are also a number of technical drawbacks associated with this traditional culture system. For example, it is nearly impossible to quantify how many cells are transferred into a small plate,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08C12N5/074
CPCC12N2501/115C12N2502/1323C12N2509/00C12N5/0606
Inventor R・斯特瑞尔C・埃勒斯特罗姆H・赛伯
Owner CELLECTIS SA
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