Method of dynamically culturing embryonic stem cells

a technology of embryonic stem cells and culturing methods, applied in the field of dynamic culturing human embryonic stem cells, can solve the problems of large cell clumps, inability to large-scale clinical production, purity and reproducibility of eb,

Inactive Publication Date: 2011-02-03
TECHNION RES & DEV FOUND LTD
View PDF32 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]According to one aspect of the present invention there is provided a method of dynamically generating embryoid bodies comprising culturing embryonic stem cells in a bioreactor selected capable of subjecting cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in the fluid to thereby obtain dynamically generated embryoid bodies.
[0014]According to an additional aspect of the present invention there is provided a method of dynamically expanding embryonic stem cells while maintaining the embryonic stem cells in an undifferentiated state, the method comprising culturing embryonic stem cells in a bioreactor selected capable of subjecting cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in the fluid to thereby obtain dynamically expanded undifferentiated embryonic stem cell cultures.
[0018]According to still further features in the described preferred embodiments the bioreactor is capable of maintaining the embryonic stem cells in a state of free fall.

Problems solved by technology

However, the extent of EB aggregation should be carefully monitored and controlled since large agglomerated EBs are often accompanied with extensive cell death and necrosis due to mass transport limitations (Dang et al., 2002).
However, although adequate for small-scale laboratory purposes these systems are not amenable to large-scale clinical production due to the inability to control the extracellular components, which may affect EB's purity and reproducibility, as well as the cell types which can be readily generated therefrom [Maltepe, E. et al., (1997).
On the other hand, prior art attempts to culture EBs in spinner flasks, as a simple, large-scale process, resulted in either the formation of large cell-clumps within a few days (Wartenberg et al., 2001), or in a massive hydrodynamic damage to the cells when extensive mixing was used (Chisti, 2001).

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method of dynamically culturing embryonic stem cells
  • Method of dynamically culturing embryonic stem cells
  • Method of dynamically culturing embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Dynamic Culture of Human Embryonic Stem Cells for the Formation of Human Embryoid Bodies

[0165]According to the current practice, the formation of embryonic bodies (EBs) involves the aggregation of multiple hES cells via a static growth of ES cultures, a procedure which results in large aggregates with necrotic centers. The following example illustrates a novel approach for dynamically generating hEBs thereby allowing large-scale production of small-size EBs suitable for therapeutic applications.

Materials and Methods:

[0166]Human ES Cell cultures—Undifferentiating hESCs [H9.2 passages 36-60 (Amit et al., 2000)] were grown on an inactivated mouse embryonic feeder layer (MEF) with culture medium consisting of 80% knockout (KO) DMEM (Gibco-Invitrogen Co.) supplemented with 20% (v / v) defined fetal bovine serum (FBSd; HyClone, Utah, USA), 1 mM L-glutamine, 0.1 mM mercaptoethanol, and 1% non-essential amino acid stock (all from Gibco-Invitrogen Co.). The hESCs were fed daily with fresh medi...

example 2

[0178]EB Formation is More Efficient in the STLV Bioreactor than in Static Cultures

[0179]To examine the efficiency of hEBs formation using the STLV bioreactor the number of hEBs and the DNA and protein contents were determined.

Materials and Methods

[0180]Medium sampling and analysis—Human ESCs were cultured using either the STLV bioreactor or the static cultures. Every third-fifth day 70% of the medium was replaced according to manufacture instructions. Shortly, vessels were stopped and aggregates were allowed to settle down. Triplicate samples from the medium were immediately analyzed for pO2, pCO2, pH, glucose and lactic acid levels using Blood Gas Analyzer, (StatProfile M, Nova Biomedical, Boston) or stored at −70° C. for analysis of lactate dehydrogenase (LDH) activity (Hitachi Modular P800, Tokyo, Japan). Glucose consumption at a given time point was determined from the medium concentration of glucose taking into account the 70% volume replacement of the medium.

[0181]Biochemical...

example 3

[0195]The STLV Bioreactor is Suitable for EB Differentiation

[0196]To further examine the suitability of the STLV and HARV bioreactors to support EB differentiation, one-month-old EBs were subjected to histological staining, immunohistochemistry and RT-PCR analyses.

Materials and Methods

[0197]Immunohistochemistry—Immunostaining was preformed following deparaffinization of histological sections using the Dako LSAB®+ staining kit for anti glial fibrillary acidic protein (GFAP), anti CD34 and somatostatin (all from Dako Corp, Carpenteria, Calif., USA) according to manufacturer's instructions. Both IgG isotypes and secondary antibody staining were used as controls.

[0198]Reverse transcription (RT)-PCR analysis—Total RNA was extracted from hEBs using the TriReagent (Sigma) according to manufacturer's instructions. To ensure no DNA contamination, RNA samples were treated with DNA free kit (Ambion Inc., Austin, Tex., USA) and examined for DNA contamination prior to performing the RT reaction....

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
volumeaaaaaaaaaa
diameter sizeaaaaaaaaaa
volumeaaaaaaaaaa
Login to view more

Abstract

The present invention is of a method of dynamically generating human embryoid bodies which can be used for generating lineage specific cells and cell lines. Specifically, the present invention can be used to generate ESC-differentiated cells for cell-replacement therapy.

Description

RELATED PATENT APPLICATION[0001]This application is a Continuation of U.S. patent application Ser. No. 10 / 536,439 filed on Nov. 28, 2005, which is a National Phase Application of PCT Patent Application No. PCT / IL03 / 01017 having International Filing Date of Nov. 30, 2003, which claims the benefit of U.S. Provisional Patent Application No. 60 / 429,574 filed on Nov. 29, 2002.[0002]The contents of the above Applications are all incorporated herein by reference.FIELD AND BACKGROUND OF THE INVENTION[0003]The present invention relates to a method of dynamically culturing human embryonic stem cells (hESCs) which method can be utilized for large scale production of embryoid bodies (EBs) and EBs-derived-cells and cell lines and thus is particularly suitable for the generation of cell cultures utilized in cell replacement therapy.[0004]Human embryonic stem cells (hESCs) are proliferative, undifferentiated stem cells capable of being maintained in an undifferentiated state while preserving their...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/073C12N5/00C12N5/02
CPCC12N5/0603C12N5/00
Inventor GERECHT-NIR, SHARONITSKOVITZ-ELDOR, JOSEPH
Owner TECHNION RES & DEV FOUND LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap