Passive Heat Transfer Based Cooling Rate Control Method And Its Application In Cell Cryopreservation

In the cooling process of cryopreservation,the cooling rate is an important factor affecting the survival rate of cells after cryopreservation and rewarming.The slow-cooling preservation of biological materials usually adopts a "two-step method".That is,the first step is to first cool the biological sample to-80 ℃ a lower temperature rate and maintain it in that temperature for a little time,then the second step,to put the biological samples directly into the liquid nitrogen tank(-196 ℃)for long-term cryopreservation.The damage that cells suffer during slow cooling is caused by the cooling rate of the cells.For existing biomaterials,cooling the biological sample to-80℃ is commonly performed using a liquid nitrogen program freezer(LNF).This method has the disadvantages.of high cost,complicated operation,high use and maintenance costs,and so on.In this paper,the biotransport and intracellular ice formation in freezing human umbilical vein epithelial cells(HUVEC)were studied.A mathematical model for the biotransport and intracellular ice formation in HUVECs were established and then cryogenically cooled.On the platform,the volume change and ice crystal formation law of HUVECs at l℃/min,10℃/min,and 20℃/min were calculated,and a large amount of data was obtained,in which,about 508 HUVECs was observed at the cooling rate of 1℃/min,about 370 of 10℃/min and about 331 of 20℃/min.And then through simulation and fitting,the biotransport and intracellular ice formation mechanism of HUVEC during freezing was obtained.Based on the passive cooling method,this paper designs,improves,and develops a Passive Cooling Devic(PCD)to operate the slow cooling of HUVECs.It has the advantages of inexpensive,simple operation,low cost of maintenance and use.During the design process,a thermodynamic mathematical model was established by establishing a phase change process during the passive cooling process.The size of the PCD structure was obtained through calculation and simulation.Then,a prototype of the PCD was produced through industrial CAD.In this paper,HUVECs were used for verification the function of the PCD,through which both 8 independent samples were used for testing in PCD method and LNF method.By measuring the temperature drop curve and comparing the cell viability,it was confirmed that the newly designed PCD could be used to deal with HUVECs in cryopreservation bags.Considering the current cryopreservation of biological samples,cryo vials is often used.In this paper,the mathematical model of the thermophysical properties of the cryovials directly placed in the box-controlled thermostat is established.By simulation and analysis of the heat distribution and temperature drop curve of the cryovials in the process of cooling,the cryovials is designed for the use directlu in PCD designed in this paper.In this article,the natural killer T(NKT)cells,which were commonly used in cell therapy,were tested by PCD and LNF through both 10 independent samples.Through the cell viability and the ability for Anti-tumor efficiency of NKT cells,it was proved that the PCD can replace the liquid nitrogen program cooler in the preservation of NKT cells which wasstored in cryovials.In this paper,we cooperated with Hebei Life Origin Bio-Technology Company to separately store HSCs and MSCs with a newly designed PCD,and compared them with LNF for simultaneous preservation.HSC and MSC cells cryopreserved by both methods had the same cell viability and cell recovery.In addition,we also tested HSC colony-forming units(CFU)to evaluate the potential of in-vitro differentiation of HSCs and the implantation potential of the graft in-vivo,and stem cell differentiation experiments of MSC cells,and demonstrated that the cells cryopreserved in both ways did not affect the basic function of both cells.In addition,the HSC-CFU test was performed at 6 months,12 months,and 18 months after HSCs were cryopreserved using PCD method.The results showed that the cells had normal function.