Cryoprotective aqueous solutions useful for the preservation of

Details Cryoprotective aqueous solutions useful for the preservation of Cryoprotective aqueous solutions useful for the preservation of

1995-06-01

1997-09-23

Richter, Johann

Chemistry: molecular biology and microbiology

Differentiated tissue or organ other than blood, per se, or...

Including perfusion; composition therefor

4352401, 435240241, 424574, A01N 100, C12N 500, A61K 3512

Patent

active

056703082

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

This application is a 371 of PCT/EP93/03364 filed Dec. 1, 1993.
The present invention refers to the preservation, by freezing, of epithelium sheets obtained from cell cultures of keratinocytes.
The cultures of human keratinocytes, developed by. Green and Rheinwald (U.S. Pat. Nos. 4,016,036; 4,304,865; 4,456,687) raise more and more interest in several medical applications such as in the treatment of serious burns, ulcers, difficult to heal scars and in plastic oncological and reconstructive surgery.
In the case of seriously burnt patients, the death-rate, depending on the patient's age and affected body surface, is dramatically high, particularly for third degree burns greater than 60%. According to the usual procedure, the damaged body surfaces are covered by transplanting skin films removed from healthy areas (auto-grafts); this procedure is very time-consuming and cannot be practically used when the burnt area exceeds 60% of the body surface. The technique by Rheinwald and Green has allowed the burnt areas to be covered with autologous epithelial films obtained from the patient's epithelial cells (keratinocytes). Starting from a small biopsy of epithelial tissue, the technique affords an epithelium surface sufficient to cover the whole body surface in a few weeks.
The autologous transplant in seriously burnt patients represents a unique, socially relevant therapeutic approach.
Autologous transplants are carried out also in cases of plastic oncological and reconstructive surgery. In this case, before surgery, sheets of epithelium expanded in vitro are prepared starting from a patient's biopsy.
The use of cultured human epithelium sheets as "living bandage", for the treatment of ulcers and scars which are difficult to heal, is particularly interesting not only from the social but also from the commercial point of view. In this case, the epidermis sheet, optionally obtained from suitably typized keratinocytes cultures, is used as a temporary dressing of the scar, making the restoring of the epithelium thereon easier thanks to the production of a series of factors stimulating the growth of this tissue, decreasing therefore the patient's suffering and hospitalization.
More than 4 million patients could be treated in the United States with this new therapeutic approach. It is obvious however that this would require remarkably reduced costs of the epithelium sheets, long and easy preservability of the product and easy application procedures in hospital environments.
The method originally developed by Green and Rheinwald for autografts comprises the sampling of a small fragment (2-3 cm.sup.2) of epithelium from a healthy area, from which an epithelial cellular suspension is obtained and cultured and finally expanded by means of subsequent passages of the primary cultures. When the secondary and tertiary cultures become confluent, multilayered epithelium sheets are obtained in different sizes according to the need.
Notwithstanding the high potentiality of this therapeutic approach, its wide-scale application is limited by the requirement that the obtained epithelial sheets should be used within a few hours after their preparation.
EP-A-0296475 discloses a method for preserving at -100.degree. C. viable, transplantable epithelium sheets cultured in vitro, frozen in a nutrient medium containing the usual cryopreserving agents such as dimethylsulfoxide or glycerine.
Although this method has allowed for the first time the preservation of in vitro cultured epithelium sheets, a number of problems limit its industrial applicability: critical for the cells at room temperature. This makes critical the initial step of the preserving process, comprising an incubation at room temperature to equilibrate the epithelium sheets with the solution and the final phase of thawing before using the sheets. In both cases, a few minutes difference in the performance of these steps may (irreparably) impair the characteristics of the product; characterized by different cooling rates in particular temperature

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Cryo-Letters, vol. 3, (1982), pp. 115-120, F. Franks, "Apparent Osmotic Activities of Water Soluble Polymers Used as Cryoprotectants".
Breda et al., Cryobiology 29, 281-90 (1992).

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