Refrigeration – Processes – Deodorizing – antisepticizing or providing special atmosphere
Patent
1997-07-30
1999-10-12
Kilner, Christopher B.
Refrigeration
Processes
Deodorizing, antisepticizing or providing special atmosphere
4352975, C12M 300
Patent
active
059640960
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the cryopreservation of both harvested tissue and cultured tissue equivalents made using in vitro technology. This invention also relates to a cryopreservation package design for both harvested tissue and cultured tissue equivalents that is both a cost effective and easy to handle package design that allows for maximal viability of the tissue or tissue equivalent to be cryopreserved. By use of the cryopreservation technology, either cryopreserved harvested tissue or cryopreserved cultured tissue may be stored for indefinite periods of time prior to use. The cultured tissue is an in vitro model of the equivalent human tissue, which, when retrieved from storage, can be used for transplantation or implantation, in vivo, or for screening compounds in vitro.
2. Brief Description of the Background of the Invention
In vitro technology has developed tissue equivalents for the purposes of in vitro testing or in vivo grafting for wound repair. Methods of producing such tissue equivalents are disclosed in U.S. Pat. Nos. 4,485,096, 4,604,346, 4,835,102 and 5,374,515 and U.S. Ser. Nos. 08/193,809 and 08/337,830; all of which are incorporated herein by reference.
The shelf life of living tissues is limited and, subsequently, their window of use is short, resulting in much waste. There is a need to preserve such tissues for extended periods of time, as for shipping and storage, until their use. Both the development of a cryopreservation method and a package for cryopreservation and storage would extend the window of use indefinitely, ease shipping and allow for the maintenance of an inventory. To enable an inventory of tissue at burn care centers and hospitals is also desirable. Other advantages are that samples can be retained from different stages of the manufacturing cycle for quality control archives and larger production batches can be made as they can be maintained in a frozen state.
Currently, the storage time of cellular biological materials is extended by cooling to "cryogenic" temperatures. The transition from the liquid into the solid state by lowering the temperature of the system can take place either as crystallization (ice), involving an orderly arrangement of water molecules, or as vitrification or amorphization (glass formation), in the absence of such an orderly arrangement of crystalline phase. The challenge for a cryobiologist is to bring cells to cryogenic temperatures and then return them to physiological conditions without injuring them.
There are two basic approaches to cryopreservation of cells and tissues: freeze-thaw and vitrification. In freeze-thaw techniques, the extracellular solution is frozen (i.e., in crystalline form), but steps are taken to minimize the intracellular ice formation. In vitrification procedures, there is an attempt to prevent ice formation throughout the entire sample. The former approach is problematic in that if ice crystals are formed inside the cells, they are detrimental to cell viability upon thawing. However, cells could survive a freeze-thaw cycle if they are cooled at controlled rates in the presence of non-toxic levels of cryoprotectants. The latter approach of vitrification seeks to avoid potentially damaging affects of intra- and extracellular ice by depressing ice formation using very high concentrations of solutes and/or polymers. However, the cell damage may occur to long exposure to toxic levels of these additives required for vitrification.
Cryoprotectants protect living cells from the stresses involved in the freezing process. One way cryoprotectants protect cells is by diluting the salt that becomes increasingly concentrated in the unfrozen solution as water is transformed to ice. The amount of ice is dictated by the temperature and initial composition of the solution; whereas the amount of unfrozen fraction is a function of temperature only. Cryoprotectants have several other functions. An important one is that they usually reduce the intracellular ice formation temperatu
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Toner Mehmet
Tschumakow Alexander G.
Watson Stephen R.
Kilner Christopher B.
Organogenesis Inc.
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