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Cryopreservation of hepatocytes

A technology of cryopreservation and hepatocytes, applied in the preservation of human or animal bodies, animal cells, vertebrate cells, etc.

Inactive Publication Date: 2008-11-26
PROMETHERA BIOSCIENCES SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Until now, there are no known cryopreservation methods by which human hepatocytes can be successfully cryopreserved in order to subsequently perform longer in vitro studies

Method used

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  • Cryopreservation of hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of human hepatocytes for cryopreservation

[0040] 1.1 Isolation of human hepatocytes

[0041] Hepatocytes from human donors are isolated in a manner known per se from tissue parts surgically removed by any means, which requires the consent of the donor. To this end, the tissue is perfused, the hepatocytes are detached from the tissue complex, and the hepatocytes can be obtained from the perfusion solution. The viability of harvested hepatocytes was determined by trypan blue staining. For further experiments, only cell preparations showing trypan blue exclusion above 70% were used.

[0042] 1.2 Preparation of matrix

[0043] In the following experiments, cell culture vessels in the form of multiwell plates, 6-well plates (type 657160, GreinerBio-One) were used. Culture plates are coated with native collagen gel, preferably isolated from rat tails. Alternatively, multiwell plates were used - 6-well plates pre-coated with type I collagen (typ...

Embodiment 2

[0054] Example 2: Thawing of Cryopreserved Hepatocytes

[0055] Hepatocyte cultures frozen according to Example 1 and stored at -151°C were thawed and re-cultured after a storage period of up to 4 weeks for further use in in vitro experiments.

[0056] For thawing of cryopreserved hepatic parenchymal cell cultures, the 6-well plates were first immediately transferred to an incubator operating under standard conditions for 5 minutes after removal from the freezer or nitrogen tank (see Example 1). Subsequently, 1 ml of solution 1 preheated to 37° C. (see Example 1) was slowly added dropwise to each well. This method was repeated for up to 3 simultaneously thawed 6-well plates, ie up to 18 wells.

[0057] Subsequently, the process was repeated, and 1 ml of solution 1 was slowly added to each well. Then, aspirate the supernatant with a Pasteur pipette. The supernatant essentially contained thawed non-viable and non-adherent cells.

[0058] For further washing, in each case, ...

Embodiment 3

[0062] Embodiment 3: the mensuration of living cell number

[0063] The number of viable (living) cells in cultures of freshly isolated hepatocytes and in cultures of cryopreserved hepatocytes according to the invention was determined by morphologically intact cell counting methods. Here, for 0.259mm 2 Photos of the size of the control area were counted. From the number of intact cells found in the control area, the number of intact cells in the entire cell culture vessel (for the 9.6 cm well used 2 area of ​​a 6-well plate, the correction factor is 3700).

[0064] Morphology of re-cultured hepatocytes was photographically documented before freezing and at various time points after thawing from cryopreservation and compared with cultures of freshly isolated and cultured hepatocytes.

[0065] result:

[0066] Figure 1 shows the morphology of human hepatocytes freshly isolated and seeded on the collagen gel layer ( Figure 1A ) and the morphology of isolated human hepatocyte...

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Abstract

The invention relates to methods for preparing hepatic cells for cryopreservation, to methods for the cryopreservation of isolated hepatic cells, and to methods for producing a culture of cryopreserved isolated hepatic cells.

Description

technical field [0001] The present application relates to the technical field of cryopreservation, in particular to methods for preparing hepatocytes suitable for cryopreservation, methods for cryopreserving isolated hepatocytes and methods for preparing sandwich cultures of isolated hepatocytes for cryopreservation . Background technique [0002] Liver cells, especially hepatocytes, isolated from tissue complexes are particularly useful in the form of primary cell cultures for testing the physiological effects of drug candidates. Primary hepatocytes, freshly prepared from humans, represent in particular the "gold standard" for the identification of candidate active substances in in vitro series or for active substance metabolism or enzymatic studies. Unfortunately, freshly isolated human hepatocytes are not routinely and readily available. Therefore, there is a need for a method that can store isolated hepatocytes for a certain period of time. Here, the physiological fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/06C12N5/071
CPCA01N1/0231A01N1/02C12N5/067C12N2533/54A01N1/0284C12N5/0602
Inventor 卢博米尔·阿尔谢尼耶夫克拉希米拉·亚历山大德罗娃马克·巴托尔德扎比内·卡费特-卡斯汀布里塔·劳贝
Owner PROMETHERA BIOSCIENCES SA
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