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Banking of multipotent amniotic fetal stem cells

Inactive Publication Date: 2005-02-24
HAAS MARTIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The invention is a cell bank system that contains preserved samples of amniotic fluid-derived cells, which can be identified and retrieved by the donor. These cells can be multipotent or pluripotent stem cells, which can be cryopreserved and stored for future use. The system also includes a database with information about the identity of the donor and the stem cells. The technical effects of the invention include the ability to preserve and retrieve viable stem cells for future use, as well as the ability to store and retrieve data relating to the identity of the donor and the stem cells."

Problems solved by technology

The political, moral and ethical issues around hES cells, as well as the perceived difficulties of expanding undifferentiated adult stem cells in culture, while maintaining a genetically normal genome, are major barriers in the development of human cell replacement therapy.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation and Expansion of Undifferentiated Cells Derived from Amniotic Fluid

[0083] Approximately 2 to 5 ml of fresh amniotic fluid was harvested from women undergoing routine amniocentesis at 16 to 21 weeks of pregnancy (2nd trimester). Second trimester amniotic fluid contained approximately 1-2×104 live cells per ml. The cells were pelleted in a clinical centrifuge and resuspended in 15 ml “MAFSC” medium. MAFSC medium was composed of low glucose Dulbecco Modified Eagle's Medium (GIBCO, Carlsbad, Calif.) and MCDB 201 medium (SIGMA, Saint Louis, Mo.) at a one to one ratio and contained 2% Defined Fetal Calf Serum (HYCLONE, Logan, Utah), 1× insulin-transferrin-selenium, linoleic-acid-bovine-serum-albumin (ITS+1, SIGMA), 1 nanomolar dexamethasone (Sigma), 100 μm ascorbic acid 2-phosphate (Sigma), 4 μm / ml gentamycin, 10 ng / ml of rhEGF (R&D Systems, Minneapolis, Minn.), 10 ng / ml rrPDGF-BB (R&D) and 10 ng / ml rhFGF-basic (R&D). The wells of 6-well culture dishes were prepared for cell pl...

example 2

Morphological Characterization of Cell Types

[0086] Cultured amniotic fluid-derived cells were tested for cell surface and differentiation markers and were karyotyped. These cells were found to be immortal or near-immortal and were named Multipotent Amniotic Fetal Stem Cells (MAFSC).

[0087] All of >80 amniotic fluid sample harvests of 5 ml gave rise to at least one adherent MAFSC colony and continuous culture. The majority of sample harvests gave rise to 3-4 individual clones. Among the individual clones, different colonies / cultures had diverse colony morphologies, as shown in FIG. 1. Some cultures had a flat, epihelial morphology (FIG. 1A). Others had a fibroblastic morphology (FIGS. 1B, 1C). Both the epithelioid and the fibroblastic classes of cultures senesced after ˜60 population doublings (PD), yielding a maximum of 1018 cells, unless the cells were immortalized by the expression of the human TERT (telomerase) gene that maintained the length of the cells' telomeres. Indeed, mor...

example 3

FACS Analysis of MAFSC Cells

[0089] Cells were prepared for FACS analysis by trypsinizing to remove them from the tissue culture flask, washing in buffer, HBSS, 2% BSA, 0.1% sodium azide, then resuspended in 100 μL of the same buffer. For intracellular antigens (i.e. Oct-4), the cells were fixed and permeablized using Beckman-Coulter IntraPrep reagents, as suggested by the manufacturer. Primary antibodies specific for the indicated cell-surface or intracellular marker were added at a 1:10 dilution and incubated for 30 minutes at room temperature, then washed. For samples using primary antibodies that were not fluorescently-conjugated, the cells were then resuspended in 250 μL of buffer and the appropriate fluorescent-labeled secondary antibody was added at a 1:250 dilution and incubated for 30 minutes at room temperature. Labeled cells were washed and resuspended in buffer or 1% paraformaldehyde for analysis by a FACSCalibur flow cytometer. The data obtained from this analysis were ...

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Abstract

Stem cells, including those designated as multipotent amniotic fluid stem cells (MAFSC) cells are found in the amniotic fluid of mammals, including humans. MAFSCs are fetal, multipotent stem cells that can be used for any desired stem cell utility, including treatment of individuals in need of tissue replacement or gene therapy. Methods of banking MAFSCs derived from the amniotic fluid cells of pregnant individuals are disclosed. Amniotic fluid-derived cells are banked for the purpose of access to transplantation antigen-compatible or syngeneic multipotent stem cells.

Description

RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 60 / 495,513, filed Aug. 14, 2003, and U.S. Provisional Application No. 60 / 495,437, filed Aug. 14, 2003, the disclosures of which are incorporated by reference herein in their entireties.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates to the field of stem cell research. Specifically, the invention relates to the preservation and banking of amniotic fluid-derived cells, or multipotent amniotic fluid-derived stem cells of individuals. The cryopreserved amniotic fluid-derived cells can be stored indefinitely. Thawed cells can be used to grow stem cell lines which can create differentiated cells, such as specific cell types or tissue types. These differentiated cells are capable of being transplanted into the individual or into unrelated matching individuals if needed. [0004] 2. Description of the Related Art [0005] Stem cells can...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N1/02C12NC12N5/073C12N5/074G01N33/48G01N33/50G06F19/00
CPCC12N5/0605C12N5/0607C12N2500/25C12N2501/39C12N2501/115C12N2501/135C12N2501/11
Inventor HAAS, MARTIN
Owner HAAS MARTIN
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