Cryopreservation of cells and subcellular fractions

a cell and subcellular technology, applied in the field of cryopreservation of self-sustaining bodies, can solve the problems of limiting the ability to conduct experiments using such a system, and affecting the viability of cells after thawing

Inactive Publication Date: 2011-05-05
XENOTECH CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, the availability of viable, fresh liver tissue from humans is inconsistent and overall fairly limited, thus limiting the ability to conduct experiments using such a system, because availability does not always coincide with when such cells are needed.
However, individual hepatocyte samples have limited applicability due to individual variation in cell function.
While the development of cryopreservation methods for the storage of hepatocytes has significantly facilitated the availability of human hepatocytes, cryopreservation has been found to cause significant decrease in cellular viability after thawing.
However, the poor recovery of cells following cryopreservation and thawing continues to limit the use of hepatocytes for in vitro liver models.
This problem is particularly apparent in traditional pooled hepatocyte preparations, which are prepared using multiple freeze-thaw cycles, where each successive freeze-thaw cycle causes increased damage to at least a portion of the hepatocytes in the preparation, reducing overall cell viability of the resulting pool.
Similar problems are encountered during cryopreservation and storage of other cellular and subcellular fractions, such as organelles.

Method used

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  • Cryopreservation of cells and subcellular fractions
  • Cryopreservation of cells and subcellular fractions
  • Cryopreservation of cells and subcellular fractions

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation of Hepatocytes

A. Reagent Preparation

[0040]Various reagents for use in the following Example were prepared. To prepare about 20 L of 1-×Perfusion Buffer 1 (1×-PB1), the following reagents were dissolved in 18 L of high purity water: 137.9 g NaCl, 7 g KCl, 3.3 g KH2PO4, 42 g NaHCO3, 19.8 g glucose, and 3.8 g ethylene glycol tetraacetic acid (EGTA). The pH was then adjusted to 7.4, as required, using 1-10 N NaOH or HCl at room temperature. Additional high purity water was added to reach a final volume of 20 L.

[0041]Next, 10 L of Perfusion Buffer 2 (PB2) was prepared by dissolving the following reagents in 9 L of high purity water: 69 g NaCl, 3.5 g KCl, 1.675 g KH2PO4, 21 g NaHCO3, 10 g glucose, 2.2 g CaCl2, and 1.45 g MgSO4. The pH was adjusted to 7.4, as required, using 1-10 N NaOH or HCl at room temperature. The final volume was adjusted to 10 L using additional water. PB2 is combined with collagenase (Worthington Biochemical Corp., Freehold, N.J.; 90 units / mL).

[0042]The de...

example 2

Preparation and Pre-Pooling of Cryopreserved Hepatocyte Pellets

A. Cleaning, Autoclaving and Assembling of the Pellet Holder

[0051]Pellet holder assembly consists of pellet holder base, pellet holder (cryopreserved pellet receptacle, see Example 3) and the lid.

[0052]1. The pellet holder base (a sturdy 96-well base made of plastic, that the pellet holder is placed on during the cryopreservation process) and lid (a molded plastic top that fits over the top of the pellet holder and pellet holder base that enables sterility during the cryopreservation process) were cleaned using mild soapy water, then rinse with tap water, followed by deionized water;

[0053]2. The pellet holder was cleaned by placing the pellet holder in a 1 L beaker and filling to the level of the holder wells with acetone;

[0054]3. The pellet holder was sonicated for ˜10-15 min;

[0055]4. The acetone was washed off with warm soapy water, and the pellet holder was rinsed first with tap water, then with deionized water;

[0056]...

example 3

Thawing and In Situ Pooling of Cryopreserved Hepatocytes

[0081]To thaw the pre-pooled pellet stacks, a cryo vial containing the selected stack was removed from the vapor phase N2 storage unit and quickly placed into a prewarmed shaking water bath (37±1° C.) so that the level of the water bath was above the high point of the top pellet in the stack. The pooled hepatocyte composition was formed in situ, as the individual pre-pooled pellets thaw and the formerly discrete, frozen suspensions mixed together into a single pooled hepatocyte composition in the cryo vial. For example, a stack of 10 discrete 100 μL pre-pooled pellets thawed into 1 mL of pooled hepatocyte composition. Once thawed, the cryo vials were quickly removed from the water bath, and their contents were gently poured into a vial containing DMEM+cryo (about 3-5 times volume of the pellet stack) and IsoPercoll (90% PERCOLL® in 10×PBS). The cryo vial was then rinsed with 1.5 mL of DMEM+, which was added to the pooled produc...

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Abstract

The invention provides cryopreserved compositions of cells, wherein the compositions are advantageously in the form of self-sustaining bodies that can be individually handled and combined independently of a container, allowing for easy customization of the eventual pooled preparation. The invention also provides pre-pooled stacks of the self-sustaining cryopreserved compositions for eventual thawing to produce pooled preparations of cells. A mold and methods for forming the self-sustaining bodies are also provided. The invention is also concerned with methods of forming pooled preparations of cells using single-cryopreserved compositions of cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims the benefit of and priority from U.S. Provisional Patent Application Ser. No. 61 / 340,259, filed Mar. 15, 2010, and U.S. Provisional Patent Application Ser. No. 61 / 256,833, filed Oct. 30, 2009, the entire disclosures of which are hereby incorporated by reference herein.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention is broadly concerned with cryopreserved self-sustaining bodies formed from compositions of cells, methods of forming the same, and methods of using the same to produced pooled preparations of cells.[0004]2. Description of Related Art[0005]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. One of the detoxifying functions of hepatocytes is to modify ammonia to urea for excretion. They are also impo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B30/06C12N5/02C40B40/02C12M1/00
CPCA01N1/0268A01N1/02A01N1/0284C12N5/067
Inventor CZERWINSKI, MACIEJ
Owner XENOTECH CALIFORNIA
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