Chemistry: molecular biology and microbiology – Apparatus – Differentiated tissue perfusion or preservation apparatus
Reexamination Certificate
1998-10-13
2001-12-04
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Differentiated tissue perfusion or preservation apparatus
C435S307100
Reexamination Certificate
active
06326188
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for preparing cryopreserved tissue for transplant into a human. The process is directed to the thawing and removal of cryoprotective agents from cryopreserved tissue. Cryoprotective agents, for example, dimethyl sulfoxide and glycerol, are removed from cryopreserved tissues including for example, cardiovascular tissue including heart valves, arteries and veins, and musculoskeletal tissue, following or simultaneously with thawing from the cryopreserved state by use of a continuous flow-through of wash-out solution using the present continuous perfusion chamber. The present continuous perfusion chamber may be either rigid or deformable, and includes an inlet port and an outlet port.
BACKGROUND OF THE INVENTION
Cryopreserved tissues are defined as tissues which have been frozen in the presence of one or more cryoprotective agents. Cryoprotective agents are defined as compounds which are used to reduce damage to cryopreserved tissues/cells during the freezing, storage, and/or thawing processes associated with cryopreservation.
A variety of tissues are preserved by cryopreservation in the presence of cryoprotective agents. Methods and protocols for the cryopreservation of human heart valves have been extensively described in the scientific literature since the early 1970's, however the method described in U.S. Pat. No. 4,890,457 is representative of these early methods. Of concern to the present invention is the removal of cryoprotective agents from such tissues following or simultaneous with thawing just prior to transplantation into a patient. Most cryoprotective agents used to protect the cryopreserved tissue during cryopreservation are harmful to tissues surrounding the implant due to time and temperature dependent chemical and physical damage and it is generally deemed important to remove these agents from the thawed tissue prior to transplantation. Cryoprotectant removal techniques that have been described include, procedures where the concentration of cryoprotectant is reduced via a timed series of additions of lower osmolality solutions, and procedures where removal is accomplished by adding the tissues to a given volume of lower osmolality solution such as described in U.S. Pat. No. 5,160,313. The approach using a series of additions of solutions to remove cryoprotectant from tissues is time consuming and requires particular attention to details such as elution time, temperature, and composition of the diluting solution. The approach using a given volume requires less attention to details such as elution time, temperature, and composition of diluting solution, however the osmotic shock to the cellular population is rapid and can lead to dramatic cell swelling and subsequent cell damage and/or death (osmotic shock). This osmotic shock may be mitigated by including impermeant solutes in the diluting solution, however, such diluting solutions typically result in a tissue containing an osmotic pressure of between 400 to 800 mOsm which is dramatically greater than the approximate 290 mOsm associated with normal tissue. The present inventive method solves the problem associated with prior art methods by providing a user-friendly, continuous-multi-step dilutional process for removing cryoprotective agents from cryopreserved tissue while maintaining the resultant osmotic pressure within an acceptable normal range. Using a wash-out solution of 280-290 mOsm/kg water results in greater osmotic shock but yields “final” tissue which is iso-osmolar. Using wash-out solution of 500-600 mOsm/kg water lessens the initial osmotic shock but results in “final” tissue which is hyperosmolar.
SUMMARY OF THE INVENTION
A continuous-multi-step dilutional process for removing cryoprotective agents from cryopreserved tissues is disclosed where tissue which has been cryopreserved in the presence of one or more cryoprotective agents is thawed and washed in a continuous perfusion chamber by flowing a wash-out solution of approximate iso-osmotic 280-290 mOsm/kg water or hyper-osmotic 550-800 mOsm/kg water through and around the tissue such that the cryoprotectant is reduced to a nontoxic level of 3.0% (volume to volume) or less in the tissue prior to transplantation. The invention includes both a continuous perfusion chamber and a method whereby the rate of flow of wash-out solution is automatically controlled by the continuous perfusion chamber and a standardized operating pressure for the invention.
The present invention is directed to a continual but slow removal of cryoprotectant from cryopreserved tissues using a continuous-multi-step dilution process where the diluting or wash-out solution is continually perfused through and around the tissue being processed such that the osmolality of the perfusing solution is gradually reduced during the washing and/or thawing process and the cryoprotectant concentration in the tissue is reduced to a level known to be nontoxic to cells present in that tissue.
The present invention is also directed to a process for simultaneously thawing the cryopreserved tissue and removing the cryoprotectants using the present and continuous-multi-step dilution process.
The present invention is directed to a continuous-multi-step dilution process which requires a minimum number of physical steps and can be used on cryopreserved tissue or thawed cryopreserved tissue.
The present invention is further directed to a continuous-multi-step dilution process which requires a minimum number of steps, minimum attention to detail by the attending personnel, and is highly reproducible.
The present invention is also directed to a continuous- multi-step dilution process which requires no monitoring of final cryoprotectant concentrations in the tissue.
The present invention is directed to a continuous perfusion chamber which when used in the continuous-multi-step dilution process, results in controlled perfusion of the tissue being processed and maintains sterility of the tissue up to the time of actual transplantation.
The present invention is directed to a single-use or multi-use rigid continuous perfusion chamber having an inlet port and outlet port.
The present invention is directed to a rigid perfusion chamber having an inlet port and outlet port where the angle of the inlet port causes a circular motion in the chamber thereby causing a stirring effect during the wash-out process.
The present invention is also directed to a deformable continuous perfusion chamber having an inlet port and an outlet port. Preferably, the inlet and outlet port are configured so as to cause a circular motion in the chamber during the continuous-multi-step dilution process, i.e. a “stirring effect.” Such configuring is readily accomplished by one of ordinary skill in the art to which the present invention pertaining without undue experimentation.
The present invention is further directed to a continuous-multi-step dilution process using the present continuous perfusion chamber where the chamber further includes a sterile basin in which the present continuous perfusion chamber is placed, where the basin is of a size sufficient such that at the end of the present process the waste wash-out solution is at a level below the outlet port of the present continuous perfusion chamber.
The present invention is directed to a continuous perfusion chamber having an inlet port and outlet port where in-flow tubing is attached to the inlet port and out-flow tubing is attached to the outlet port.
The present invention is directed to a continuous perfusion chamber having an inlet port and an outlet port where sterile in-flow tubing is attached to the inlet port and no tubing is attached to the outlet port.
The present invention is also directed to a deformable continuous perfusion chamber having an inlet port and an outlet port where the deformable continuous perfusion chamber is placed in a sterile basin using sterile means such that the deformable perfusion chamber is at a level in the basin such that at the end of the continuous-multi-step dilutional process, was
Lange Perry
Wolfinbarger, Jr. Lloyd
Beisner William H.
Hopkins Susanne M.
Lifenet
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