A method and system for cryopreservation to achieve uniform viability and biological activity

Abstract

A method and system for controlled rate freezing and nucleation of biological materials is provided. The presently disclosed system and method provides the ability to rapidly cool the materials contained in vials or other containers within a cooling unit via forced convective cooling and optionally simultaneous pressure drop using uniform and unidirectional flow of cryogen in proximity to the plurality of vials disposed within a cooling unit. The rapid cooling of the biological materials is achieved by precisely controlling and adjusting the temperature of the cryogen being introduced to the system as well as the chamber pressure as a function of time.

1 . A method of controlling a chilling or freezing process of biological material disposed in a plurality of containers, comprising the steps of:
 (i) placing said plurality of containers of said biological materials in a cooling area defined as an area between a gas distribution surface and a parallel gas collection surface within a cooling chamber;   (ii) mixing a liquid cryogen with a warmer gas to produce a cold cryogenic gas at a selected temperature profile, said temperature profile corresponding to a desired cooling rate of said biological materials within said containers;   (iii) delivering a unidirectional flow of said cold cryogenic gas through the gas distribution surface to said cooling area between said parallel gas distribution and said gas collection surfaces and generally parallel to each of said plurality of containers to uniformly cool said biological materials within said containers; and   (iv) promptly exhausting said gas from said cooling chamber via said gas collection surface so as to prevent recirculation of said gas within said cooling area;   wherein greater uniformity of said biological materials in each of said plurality of containers is achieved.   
     
     
         2 . The method of  claim 1 , wherein said collection surface and/or said gas distribution surface are porous surfaces. 
     
     
         3 . The method of  claim 1 , wherein said step of mixing liquid cryogen with said warmer gas further comprises mixing liquid nitrogen with either room temperature nitrogen gas from a nitrogen supply source or with recycled gas exiting from the cooling chamber or a combination thereof and said mixing occurs in cryogen intake circuits to produce a cold nitrogen gas at said selected temperature profile, said temperature profile corresponding to a desired cooling rate of said biological materials within said containers. 
     
     
         4 . The method of  claim 1  wherein the step of delivering a unidirectional flow of said cold cryogenic gas further comprises performing a temperature quench of said biological material in each of said plurality of containers by delivering a unidirectional flow of cold cryogenic gas having a temperature of 40° C. or more below the temperature of said biological material in said plurality of containers to induce nucleation of freezing in said biological materials. 
     
     
         5 . The method of  claim 1  further comprising the step of rapidly reducing the pressure in the cooling chamber during the step of delivering a unidirectional flow of said cold cryogenic gas to induce nucleation of freezing in said biological materials. 
     
     
         6 . The method of  claim 1  wherein greater uniformity of said biological materials further comprises greater uniformity of the cell viability of said biological materials in said plurality of containers. 
     
     
         7 . The method of  claim 1  wherein greater uniformity of said biological materials further comprises greater uniformity of the biological activity of said biological materials in said plurality of containers. 
     
     
         8 . The method of  claim 1 , wherein said desired cooling rate of said biological materials within said containers is between about −2.5° C./min to about −5.0° C./min. 
     
     
         9 . The method of  claim 1 , wherein said plurality of containers comprise at least 10,000 vials. 
     
     
         10 . The method of  claim 1 , wherein said plurality of containers comprise at least 50,000 vials. 
     
     
         11 . The method of  claim 1 , wherein said plurality of containers comprise a plurality of bags. 
     
     
         12 . The method of  claim 1 , wherein said biological material in each of a plurality of containers comprises; microorganisms, tissues, organs, stem cells, primary cells, cell lines, small multicellular organisms, complex cellular structures, live or attenuated viruses, nucleic acids, monoclonal antibodies, polyclonal antibodies, biomolecules, non-peptide analogues, peptides, proteins, RNA, DNA, oligonucleotides, and/or viral particles. 
     
     
         13 . A cooling unit, comprising a uniform flow cryogenic chiller including a cryogen intake circuit coupled to a source of cryogen wherein said cryogenic chiller further includes a base gas injection box, a porous metal plate disposed or set in or near the top surface of said gas injection box, and a corresponding gas removal box positioned immediately above said base gas injection box with said porous metal plate disposed therein. 
     
     
         14 . A cooling unit comprising a chilling or freezing control system for controlling a cryogen source, an intake circuit coupled to said cryogen source and adapted for providing a uniform flow and temperature of a cryogenic cold gas to said cooling chamber, and wherein said cooling unit also comprises an intake plenum, an exhaust manifold, and two or more parallel porous surfaces that define a cooling area between adjacent parallel surfaces with one of said parallel porous surfaces disposed adjacent to said intake plenum and in fluid communication with said intake plenum and another of said parallel porous surfaces disposed adjacent to said exhaust manifold, said parallel porous surfaces and associated cooling area adapted to retain, or hold, a plurality of containers of biological materials.