Composition for cryopreservation of cells and tissues

a technology for cells and tissues, applied in the field of agents for cryopreservation of human and animal cells and tissues, can solve the problems of deterioration of cell starting performance, inability to meet certain kinds of cells, and high demand for new cryopreservation materials with low toxicity, so as to achieve the effect of curbing the deterioration of the starting performan

Inactive Publication Date: 2011-07-14
BIOVERDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025]Non-freezing agents according to the invention is also applicable in industrial-use fuel cells as to curb deterioration of their starting-up performance due to freezing of liquid.

Problems solved by technology

Conventional cryopreservation methods including a rapid freezing technique cannot preserve the complete structural integrity of cells or tissues after freezing and thawing; therefore, new cryopreservation materials with low toxicity are greatly demanded.
Moreover, DMSO is known to induce differentiation of cells such as HL-60 cells; thus, it is not suited for certain kinds of cells.
Anti-freeze proteins and glycoproteins have excellent preserving capabilities, but are too costly (JPY1,300,000YEN / g) to be used for food materials, not to mention, for cells and tissues.

Method used

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  • Composition for cryopreservation of cells and tissues
  • Composition for cryopreservation of cells and tissues
  • Composition for cryopreservation of cells and tissues

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Cryopreservative Solution

[0042]A 25% aqueous solution of ε-poly-L-lysine (made by Chisso Corporation; Molecular weight: 4000) was used; and a 20% aqueous solution of polyarylamine (Nittobo, molecular weight 5000 [PAA-05L], 15000 [PAA-L], 60000 [PAA-H]) was used. Each of the solution is added with 0-100 mol % succinic anhydride (Wako Pure Chemical Industries) on basis of amino groups of the polyamine polymer to obtain poly-amines having blocked amino groups with different amino-groups-blockage rates. Each poly-amine solution was added to Dulbecco's Modified Eagle Medium (DMEM, Sigma Aldrich) by 0-10 w / w %. On this occasion, pH of the medium was adjusted to 7.0-8.0 with 1N hydrochloric acid or sodium hydroxide solution. Further, the osmotic pressures of the media were measured by a vapor pressure osmometer (Type 5520, Wescor) and adjusted with 10% sodium chloride aqueous solution.

example 2

Cryopreservation of Cultured Cells

[0043]In a cryovial (Simport Plastics), 1×106 cells of each of cell species of L929, MG63, Caco-2 (Japan Sumitomo Pharmaceuticals), Colon26, HT1080, B16F1 and KB cell (ATCC) are suspended in 1 mL of each cryopresevation liquid; and then were frozen in a −80° C. freezer. After one week, the cells were quickly thawed in a 37° C. water bath, washed in DMEM and subjected to cell mortality test with trypan blue dye. The thawed cells were then seeded in 6-well culture plates at 1×105 cells / well, and cell survival rate was evaluated with trypan blue dye after 6 and 24 hours of culturing. A commonly-used cryopreservative, which is 10% DMSO in fetal bovine serum (FBS), was used as a cryopresercation liquid of comparative example.

[0044]As shown in FIG. 1, when used as the cryopreservation liquid in cryopreserving L929 cells was each 7.5% solution of the poly-lysine (PLL) having been modified by adding 50% or more molar amount of succinic anhydride on basis of...

example 3

Toxicity Test

[0048]Toxicity test was performed on L929 cells. The cells having been suspended in a culture medium of DMEM with 10% fetal bovine serum are seeded in 96-well plates (1.0×103 cells / well) and cultured at 37° C. for 72 hours. Thereafter, each of ε-poly-L-lysine and the modified poly-lysines having been added with varying concentrations of succinic anhydride was added to the culture media to attain final concentrations of 0-10%. Then, after the culture for 48 hours, concentration values at 50% cell growth inhibition were measured as IC50 by MTT assay, relative to cell growth in the culture medium not added with the polymer. Table 2 shows the results; and a preservation liquid of comparative example is the DMSO solution (10% DMSO / fetal bovine serum). As seen from Table 2, IC50 values for the PLLs succinc anhydride 58%, 63% and 79% were 2-3 times of that for the DMSO solution; this indicates that the toxicity of the poly-lysine is ½-⅓ of that of the cryopreservation liquids ...

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Abstract

A composition for cryopreservation of cells and tissues of human and other animals in a safe manner without using toxic substances such as DMSO, as well as for freeze preserving or freeze-drying of foods and pharmaceuticals. In examples, ε-poly-L-lysine is reacted with succinic anhydride so that 60% or more of amino groups are blocked; and, thus obtained polymer compound is added to Dulbecco-modified eagle MEM culture medium (DMEM) on market sale to form a cryopreservation liquid. In examples for foods or pharmaceuticals, the ε-poly-L-lysine derivative was added by 0.5-10 wt % to curb freeze concentration.

Description

FIELD OF THE INVENTION[0001]The invention relates to an agent for cryopreservation of human and animal cells and tissues, which is able to alleviate damages or injuries on the cells and tissues at the time of freezing and thawing the same. This cryopreservation technology is expected to be highly demanded in transplantation medicine where living tissues such as the skin, cornea, pancreatic islets and heart valves need to be cryropreserved, and in regenarative medicine where cells such as hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, iPS cells (induced pluripotent stem cell) or the like need to be cryopreserved.BACKGROUND OF THE INVENTION[0002]Cryopreservation techniques at temperatures at or below 0° C. are routinely used for long-time preservation of water-bearing or aqueous materials such as cells and tissues of plants and animals as well as foods. It is known that upon freezing these materials, ice crystals form, resulting uneven concentrations of solute...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N25/00C12N5/071A01P15/00C09K15/20C09K15/18A23L1/305H01M2/14B05D5/00
CPCA01N1/0221C12N5/0602A61K47/34A23L3/375A61K9/19A01N1/02C12N5/00C12N5/04C12N5/06A23V2002/00
Inventor MATSUMURA, KAZUAKISUGAI, HAJIMEHYON, SUONG-HYU
Owner BIOVERDE
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