In vitro techniques for use with stem cells

A technology of cells, progenitor cells, applied in the field of in vitro technology using stem cells

Active Publication Date: 2012-05-30
KWALATA TRADING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isolation of progenitor cells from BM containing multiple cell types is likewise more technically challenging

Method used

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  • In vitro techniques for use with stem cells
  • In vitro techniques for use with stem cells
  • In vitro techniques for use with stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0478] Example 1: Coating T-75 flasks with autologous plasma

[0479] For 20 T-75 flasks: ready on the day of cell preparation

[0480] Coating of T-75 flask surfaces with plasma can be performed using autologous plasma taken from centrifuged tissue samples, or plasma from a different source, such as commercially available plasma from Chemicon of Temecula, CA, US, can be used.

[0481] Plasma was collected from the upper fraction of Ficoll tubes.

[0482] Each flask was filled with 2-5 ml plasma.

[0483] Incubate at 37°C for at least 30 minutes.

[0484] Clear plasma.

[0485] Wash the flask twice in 10 ml of PBS.

[0486] The flask is ready to use.

Embodiment 2

[0487] Example 2: Coating of T-75 flasks with 25 μg / ml of fibronectin

[0488] For 20 T-75 flasks: (a) prepared the day of cell preparation, or (b) prepared the day before cell preparation.

[0489] Prepare 50 ml of 25 μg / ml fibronectin solution in PBS

[0490] Add 250μl fibronectin 5mg / ml to 50ml PBS

[0491] Fill each flask with 2-5 ml of 25 μg / ml fibronectin.

[0492] Incubate at 37°C for at least 30 minutes.

[0493] Collect the fibronectin solution.

[0494] Fibronectin solutions are reusable if stored at 4°C in sterile 50ml tubes, typically for up to 1 week.

[0495] Wash the flask twice in PBS.

[0496] Dry the flask at room temperature.

[0497] Flasks can be stored dry at room temperature (RT) for one week.

Embodiment 3

[0498] Example 3: Coating of T-75 flasks with fibronectin and 5 μg / ml anti-CD34:

[0499] For 20 T-75 flasks: (a) prepared the day of cell preparation, or (b) prepared the day before cell preparation.

[0500] Flasks were coated with fibronectin as described in Example 1.

[0501] Prepare 25 ml of 5 μg / ml anti-CD34 solution in PBS

[0502] Add 125 μl of 1 mg / ml anti-CD34 to 25 ml of PBS

[0503] Each flask was filled with 5 μg / ml anti-CD345ml.

[0504] Incubate for 2 hours at 37°C or overnight at 4°C.

[0505] Withdraw the antibody solution.

[0506] Wash the flask twice in PBS.

[0507] The flask is spare.

[0508] cell preparation

[0509] Mix the blood by gently inverting the blood bag up and down.

[0510] Transfer the blood from the bag to a sterile 500ml bottle.

[0511] Load blood into a Ficoll gradient.

[0512] Fill the tube to 50ml with blood.

[0513] Tubes were rotated at 1050 g for 20 minutes at room temperature without interruption.

[0514] Most of ...

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Abstract

A method is provided for use with extracted blood, including (a) applying blood to a first gradient suitable for selecting first-pass cells having a density less than 1.077 g / ml; (b) applying the first-pass cells to a second gradient suitable for selecting second-pass cells having a density between 1.055 and 1.074 g / ml; (c) increasing the number of cells having a density between 1.055 and 1.074 g / ml, by culturing the second-pass cells for a period lasting between 3 and 30 days; and (d) identifying endothelial progenitor cells in the cultured cells. Other embodiments are also described.

Description

[0001] Cross Reference Related Applications [0002] This application claims the following priority: [0003] (a) U.S. Provisional Patent Application 60 / 576,266, filed June 1, 2004, entitled "In vitro techniques for use with stem cells," and [0004] (b) US Provisional Patent Application 60 / 588,520, filed July 15, 2004, entitled "Indications for stem cell use." [0005] Both applications are assigned to the assignee of the present patent application and are incorporated herein by reference. field of invention [0006] The present invention relates generally to methods and devices for treating human patients suffering from vascular disorders, and more particularly to methods and devices for promoting angiogenesis and / or neovascularization and / or vasculogenesis. Background of the invention [0007] Some arterial dysfunction results from narrowing of the arteries by fatty deposits or other vascular abnormalities. This can interfere with blood flow and / or prevent tissues and ...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N5/00
Inventor瓦伦丁·富尔加耶尔·波拉特丹尼·贝尔金达夫纳·希莫尼-扎尔克斯韦特兰娜·波罗佐夫
OwnerKWALATA TRADING