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Method for the cryopreservation of cells, artificial cell constructs or three-dimensional complex tissues assemblies

A three-dimensional composite and artificial cell technology, which is applied in the preservation of human or animal bodies, tissue culture, animal cells, etc., can solve the problems of lack of mechanical stability and negative impact on cell vitality, and achieve the effect of improving survival rate and vitality

Inactive Publication Date: 2012-01-11
NATURIN GMBH & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] However, a disadvantage of these collagen-based hydrogels is that they are usually thicker than 150 μm, which negatively affects cell viability after thawing, for reasons explained above
Furthermore, pure collagen hydrogels do not possess sufficient mechanical stability to allow controlled transfer of constructs into the body after thawing.

Method used

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  • Method for the cryopreservation of cells, artificial cell constructs or three-dimensional complex tissues assemblies
  • Method for the cryopreservation of cells, artificial cell constructs or three-dimensional complex tissues assemblies
  • Method for the cryopreservation of cells, artificial cell constructs or three-dimensional complex tissues assemblies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0165] Example 1. Preparation of collagen cell carrier

[0166] 1.1 Production of planar film of the present invention

[0167] 50.0 kg of bovine hide splits were mechanically pre-cut and washed three times with water (3×50 kg). Subsequently, the raw material was placed in 50 kg of aqueous suspension (pH value of 11.8) of 0.29 kg of calcium hydroxide for alkali treatment for 150 hours. The alkaline treatment was terminated by adding hydrochloric acid (10% by weight in water) until the pH of the suspension reached 0.6. The reaction mixture was then rinsed again with water until the suspension reached pH 2.8. The resulting "collagen shell" is then mechanically processed under temperature control (<24°C) into a gelatinous viscoelastic mass by coarse grinding and pressing the broken material through a series of perforated discs with successively decreasing pore sizes. accomplish. The yield was 78.1 kg of "concentrated" collagen mass.

[0168] 60.0 kg of said "concentrated" ma...

Embodiment 2

[0184] Example 2: Cryopreservation of adherent primary cardiomyocytes

[0185] Successful cryopreservation of cultured stem cells and / or primary cardiac cells such as cardiomyocytes is a prerequisite for future cell-based therapies in the field of cardiovascular diseases.

[0186] Cryopreservation of adherently cultured primary cardiomyocytes on biocompatible scaffolds offers several advantages in terms of cell survival, cell organization, and cell biological function compared to freezing single-cell suspensions.

[0187] To assess cryopreservation of adherent primary cardiomyocytes, cardiac cells were isolated from fetal mice (E15) and cultured according to Example 1 (see Figure 5 ) prepared on the collagen cell carrier. Subsequently, the cell-seeded scaffolds were frozen in liquid nitrogen for 3 weeks and analyzed after thawing.

[0188] Fetal mouse hearts were prepared and collected in Hanks' balanced buffered salt solution (PAA, Pasching, Austria). Ventricles were isol...

Embodiment 3

[0192] Example 3: Long-term cryopreservation of adherent SAOS-2 cells

[0193] To analyze the long-term cryopreservation of adherent cells, the osteosarcoma cell line SAOS-2 was cultured on the collagen cell carrier prepared according to Example 1 and stored in liquid nitrogen for 230 days. After thawing, adherent cells were analyzed by cell proliferation assay.

[0194] Seed the SAOS-2 cells on the collagen cell carrier described in Example 1 at a density of 25,000 cells per square centimeter, in a humid incubator at 37°C and 5% CO 2 conditions for 3 days. The medium consisted of HEPES-buffered DMEM medium (PAA) supplemented with penicillin / streptomycin and 10% (v / v) FCS. For cryopreservation, these cell-seeded scaffolds were transferred to cryovials and the culture medium was replaced with freezing medium (DMEM medium buffered with HEPES supplemented with penicillin / streptomycin, 20% (v / v ) FCS and 10% (v / v) DMSO (Sigma)). The cryovials containing the collagen cell carri...

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Abstract

The present invention belongs to the field of cryopreservation of cells and tissue cultures, more specifically it refers to a method for the cryopreservation and long term storage of cells, cell constructs or three-dimensional complex tissues assemblies. The methods are based on the use of a collagen cell carrier having a specific composition as well as to a very specific thickness and appropriate mechanical properties which are maintained after thawing. The use of such collagen cell carrier (CCC) provides a very suitable support for the cryopreservation resulting in very high survival rates after thawing and providing cells and tissue assemblies already adhered to a mechanically stable and biocompatible support. The invention also refers to the frozen collagen carrier-cells assembly and to the frozen artificial cell constructs or three-dimensional complex tissue assemblies obtainable by the method of the invention and to the use thereof after thawing.

Description

technical field [0001] The present invention relates to the field of cryopreservation of cells and tissue cultures, and more particularly to methods of cryopreservation and long-term preservation of cells, cell constructs or three-dimensional composite tissue assemblies. The method is based on a collagen cell carrier with a specific composition as well as a highly specific thickness and appropriate mechanical properties, which retain these properties after thawing. The use of this type of collagen cell carrier (CCC) provides a very suitable support for cryopreservation with very high viability after thawing and provides a mechanically stable and biologically Cell and tissue assemblies on compatible supports. [0002] The present invention also relates to frozen collagen carrier-cell assemblies and frozen artificial cell constructs or three-dimensional composite tissue assemblies obtainable by the method of the present invention, and their use after thawing. Background techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCA01N1/0231C12N5/0657C12N2533/54
Inventor 蒂莫·施米特洛塔尔·贾斯特弗朗茨·马泽尔霍尔格·贝克
Owner NATURIN GMBH & CO
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