Novel Cellular Compositions and Methods for Their Preparation

a cell composition and cell technology, applied in the field of new cell compositions, can solve the problems of erratic source, limited availability of hepatocytes, development of bioartificial livers,

Inactive Publication Date: 2010-03-25
IN VITRO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A limiting factor in the development of such model systems and to the development of Bioartificial Livers (BALs) has been the erratic source and limited availability of hepatocytes, especially human hepatocytes.
The supply of such tissue is inconsistent and often geographically inconvenient in light of the limited functional lifespan of liver tissue (Smrzova, J. et al.
The poor recovery of cells when cryopreserved continues to limit the use of hepatocytes in in vitro liver models.
A second major problem affecting the use of both fresh and cryopreserved hepatocytes is the variation of liver enzyme expression that is observed in tissue from different donors (Li. A. P. et al.
However, the frequency of receipt of fresh tissue and the need to cryopreserve hepatocytes immediately after isolation has been cited as preventing the preparation of hepatocyte pools.
This difficulty remains even though pooled cryopreserved human hepatocytes are a valid model for metabolic studies (Zhang, J. G. et al.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Refreezing of Thawed Hepatocyte Preparations

[0051]Cryopreserved hepatocytes are thawed and refrozen as indicated below.

[0052]Materials: 30 cc syringe, two couplers, PERCOLL® in a 1 L bag, INVITROGRO™ CP medium in a 2 L bag, 1-2 L autoclaved beaker, heater, waterbath, centrifuge tube rack.

[0053]Procedure:

[0054]1) Set up a recirculating waterbath heater at 37-42° C.

[0055]2) Add approximately 200-400 mls of INVITROGRO™ CP medium to a 1-2 L beaker. Equip Biological Safety Cabinet with a manual pipet or a liquid handling robot.

[0056]3) Remove approximately 50 cryovials from dewar receptacle and quickly place them in 2 test tube racks. When possible, space vials apart.

[0057]4) Submerge solid cell suspensions into heated waterbath until the ice chunks can be dislodged when the vial is inverted.

[0058]Pour cell suspension from each vial into the beaker. Add 1 ml of INVITROGRO™ CP medium from the small beaker into each vial to rinse and pour contents into the beaker. Transfer thawed cell susp...

example 2

Variation of Primary Hepatocyte Samples

[0065]To illustrate the sample-to-sample variation of different sources of individual (unpooled) hepatocytes, hepatocytes are isolated from 82 different donors and analyzed for cell viability and enzyme function. The following metabolic activities are evaluated: COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST, TOLB, PHEN, and CZX. The results are shown in Table III.

TABLE IIIVariation of Hepatocyte Samples7-7-Lot No.Sex% VCOUMDEXECODHCGHCSMEPHTESTTOLBPHENCZX067M62%6717023124244352724086F74%51231050913813BQL18089F77%2521823611113BQL29090F74%3025713BQL21915BQL16091F73%132966441012252362736094F67%41123724421126401987099F86%211574BQL16010BQL22104M81%6321442472525837820105M67%5915243814129271227110F77%4524352346206115436111F71%410923314721919114F75%392321105559TBD345122M79%26302980514223425129F90%424276718116331051ACUF81%53825741888016423AITF83%45291311814BQL8215117AOKM73%60215828364586302421ATRM73%71113911BQL11834AVFM70%59135021029BQL54371124BTPM88%662936...

example 3

Characterization of Pooled Hepatocytes

[0066]Cryopreserved pooled lots of hepatocytes are prepared and analyzed for post-thaw viability and enzyme function. The following metabolic activities are evaluated: COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST, TOLB, PHEN, and CZX.

[0067]Six lots of pooled hepatocytes, comprising either five-donor pools or ten-donor pools, are prepared as described above. Hepatocytes are harvested from individual donors and then cryopreserved as individual lots using liquid nitrogen as freezing agent. Cryopreservation is accomplished by suspending the hepatocytes into freezer-safe vials containing a medium having approximately 10% DMSD and approximately 90% Cryopreservation Medium. The dispensed hepatocytes are then frozen in a controlled rate freezer until a final temperature of approximately −80° C. is reached.

[0068]To form the pooled hepatocyte preparations, individual lots are thawed, and the viable cells are isolated by PERCOLL® gradient centrifugation. Vial...

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PUM

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Abstract

The present invention relates to novel cell (e.g., hepatocyte, etc.) compositions and methods for their preparation and use. In particular, the invention concerns methods of processing preparations of such cells so as to permit their repeated cryopreservation and thawing while retaining substantial viability. The invention also concerns preparations of cells (e.g., hepatocytes) that have been repeatedly cryopreserved and thawed.

Description

[0001]This application is a continuation of U.S. patent application Ser. No. 11 / 110,879, filed Apr. 21, 2005, which application is herein incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to novel cell (e.g., hepatocyte, etc.) compositions and methods for their preparation and use. In particular, the invention concerns methods of processing preparations of such cells so as to permit their repeated cryopreservation and thawing while retaining substantial viability. The invention also concerns preparations of cells (e.g., hepatocytes) that have been repeatedly cryopreserved and thawed.BACKGROUND OF THE INVENTION[0003]Hepatocytes are parenchymal liver cells, and make up 60-80% of the cytoplasmic mass of the liver. Hepatocytes play a key role in the detoxification, modification and excretion of exogenous and endogenous substances (Ponsoda, X. et al. (2004) “Drug Metabolism By Cultured Human Hepatocytes: How Far Are We From The In Vivo Re...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N1/02C12Q1/02C12N5/071
CPCC12N5/067
Inventor DRYDEN, DANIELHARDY, JAMES
Owner IN VITRO TECH
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