Composition for cryopreservation of cells and tissues

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...

AI Technical Summary

Benefits of technology

[0018]Present invention is to provide a cryopreservation agent having excellent protective effects and low toxicity for cells or tissues, thus to replace DMSO. The present invention is also to provide an inexpensive and safe cryopreservation agent having a property similar to that of antifreeze proteins and glycoproteins to prevent freeze condensation, thus to enable cryopreservation and lyophilization of materials such as foods and pharmaceutical products.

[0020]Various animal cells including human cells, and plant cells are able to be preserved with keeping their survival rate and bioactivity are without using highly toxic DMSO or other conventional cryopreservation agent when the cells are immersed in the cryopreservation liquid and then cryopreserved at −80° C. or under cooling with liquid or vapor nitrogen. Because conventional cryopreservation agents such as DMSO, glycerin, ethylene glycol or the like are not used, toxicity upon the cells are kept to be low and the cells are able to be cryopreserved for an extended period of time without decreasing the cells' bioactivity. Further, the cryopreservation liquid is devoid of protein ingredients such as fetal bovine serum and albumin, therefore, is free of worry of infectious diseases and is not affected by lot-to-lot variations that are occasionally found in pharmaceutical products made from biological materials.

[0021]Polyamines having side-chain amino groups, such as ε-poly-L-lysine and polyallylamine, have an affinity with cell membranes due to the side-chain amino groups, thus, is considered to have cell-protecting effects. Polymer compounds having abundant carboxyl groups also have high affinity with water, thus, would help remove intracellular water to the surrounding medium on course of freezing and thereby are expected to have cryopreservation effects. Polymer compounds having both of the amino and carboxyl groups in an adequate ratio are expected to have further improved cryopreservation effects on the cells at a time of freezing. Thus, the invention is to provide a cryopreservation liquid having high effectiveness and high safety, by earnestly investigating conditions or requirements for polymer compounds having cationic groups such as side-chain amino groups, such as poly amino acids, or for polymer compounds having anionic groups such as carboxylic groups as well as for polymer compounds having both of cationic and anionic groups.

[0023]According to the invention, cryopreservation of cultured cells for experimental use would be made in a stable manner; and moreover, expected to be enabled is preservation by keeping cell functions, of functional cells such as pancreatic islets and stem cells such as ES cells, mesenchymal stem cells and iPS cells. Thus, efficiency in transplantation of these cells is expected to be improved.

[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.

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