CN100462004C - Program-controlled cooling device for cryoprotectant solution for vitrification preservation - Google Patents
Program-controlled cooling device for cryoprotectant solution for vitrification preservation Download PDFInfo
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- 239000002577 cryoprotective agent Substances 0.000 title claims abstract description 52
- 238000001816 cooling Methods 0.000 title claims abstract description 20
- 238000004017 vitrification Methods 0.000 title claims abstract description 10
- 238000004321 preservation Methods 0.000 title claims abstract description 8
- 229910001220 stainless steel Inorganic materials 0.000 claims abstract description 28
- 239000010935 stainless steel Substances 0.000 claims abstract description 28
- 238000005057 refrigeration Methods 0.000 claims abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 23
- 238000005485 electric heating Methods 0.000 claims description 11
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 abstract description 12
- 230000008014 freezing Effects 0.000 abstract description 12
- 239000012620 biological material Substances 0.000 abstract description 11
- 238000010438 heat treatment Methods 0.000 abstract description 6
- 238000003756 stirring Methods 0.000 abstract description 6
- 230000009471 action Effects 0.000 abstract description 2
- 238000005138 cryopreservation Methods 0.000 abstract description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 abstract 2
- 239000002826 coolant Substances 0.000 abstract 1
- 230000007423 decrease Effects 0.000 abstract 1
- 229910052697 platinum Inorganic materials 0.000 abstract 1
- 238000000034 method Methods 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000004804 winding Methods 0.000 description 1
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Abstract
本发明公开了一种用于玻璃化保存的低温保护剂溶液程控冷却装置。由纯低温保护剂储槽、恒流泵、存放低温保护剂溶液的不锈钢容器、电磁搅拌装置、制冷装置、加热装置、可编程控制器等组成。恒流泵不断将纯低温保护剂注入低温保护剂溶液存放容器，低温保护剂溶液在制冷装置提供的冷却介质的作用下温度不断降低，可编程控制器根据铂电阻测得的温度信号控制加热装置提供的加热量，从而使得低温保护剂溶液的温度始终高于溶液冻结点5～10℃。本发明可应用于生物材料的低温保存。
The invention discloses a program-controlled cooling device for cryoprotectant solution used for vitrification preservation. It is composed of pure cryoprotectant storage tank, constant flow pump, stainless steel container for storing cryoprotectant solution, electromagnetic stirring device, refrigeration device, heating device, programmable controller, etc. The constant flow pump continuously injects the pure cryoprotectant into the storage container of the cryoprotectant solution, and the temperature of the cryoprotectant solution decreases continuously under the action of the cooling medium provided by the refrigeration device, and the programmable controller controls the heating device according to the temperature signal measured by the platinum resistance The amount of heating provided so that the temperature of the cryoprotectant solution is always 5-10°C higher than the freezing point of the solution. The invention can be applied to cryopreservation of biological materials.
Description
技术领域 technical field
本发明涉及一种用于玻璃化保存的低温保护剂溶液程控冷却装置。The invention relates to a program-controlled cooling device for cryoprotectant solution used for vitrification preservation.
背景技术 Background technique
生物材料的低温保存已经在临床医疗和生物医学研究中得到广泛应用。在-80℃或-196℃生物材料可以保持活性几个月或几十年以上。为了避免生物材料在从室温到低温和从低温到室温的过程中受到伤害，人们使用低温保护剂(如甘油、二甲亚砜)来提供保护。在生物材料从室温降到低温前用低温保护剂溶液处理，从低温恢复到室温后再洗脱低温保护剂。使用高浓度低温保护剂可以避免冷冻过程中形成冰晶以及冰晶形成带来的各种危害，这种方法称为玻璃化法，高浓度的低温保护剂溶液也称为玻璃化溶液。Cryopreservation of biological materials has been widely used in clinical medicine and biomedical research. Biomaterials can remain active for several months or decades at -80°C or -196°C. In order to avoid damage to biological materials during the process from room temperature to low temperature and from low temperature to room temperature, people use cryoprotectants (such as glycerol, dimethyl sulfoxide) to provide protection. The biological material is treated with a cryoprotectant solution before cooling from room temperature to low temperature, and the cryoprotectant is eluted after returning from low temperature to room temperature. The use of high concentrations of cryoprotectants can avoid the formation of ice crystals during freezing and various hazards caused by ice crystal formation. This method is called vitrification, and high concentrations of cryoprotectant solutions are also called vitrification solutions.
在处于溶液状态时，低温保护剂对组成生物材料的基本单元细胞有毒性作用，该作用与以下几个因素有关：1)低温保护剂和细胞的类型；2)温度，温度越高毒性越强；3)低温保护剂浓度，浓度越高毒性越强。为了降低毒性作用，需要在较低温度下用低温保护剂溶液处理生物材料，但温度太低也会导致溶液冻结、影响低温保护剂的均匀扩散。When in solution, cryoprotectants have a toxic effect on the basic unit cells that make up biological materials, which is related to the following factors: 1) the type of cryoprotectant and cells; 2) temperature, the higher the temperature, the stronger the toxicity ; 3) The concentration of the cryoprotectant, the higher the concentration, the stronger the toxicity. To reduce toxic effects, biological materials need to be treated with cryoprotectant solutions at lower temperatures, but temperatures that are too low can also cause the solution to freeze, affecting the uniform diffusion of cryoprotectants.
发明内容 Contents of the invention
本发明的目的是提供一种用于玻璃化保存的低温保护剂溶液程控冷却装置。The object of the present invention is to provide a program-controlled cooling device for cryoprotectant solution used for vitrification preservation.
用于玻璃化保存的低温保护剂溶液程控冷却装置中的纯低温保护剂储槽与恒流泵、微型电磁阀、不锈钢容器相连接，不锈钢容器外壁设有冷却盘管、电加热丝，冷却盘管与制冷装置相连接，在不锈钢容器内壁设有温度传感器，在不锈钢容器底部设有电磁搅拌子，在不锈钢容器下方设有永磁铁，永磁铁与电机、电机调速电源相连接，电加热丝与可控硅电路、可编程控制器相连接，恒流泵、微型电磁阀、温度传感器分别与可编程控制器相连接。The cryoprotectant solution for vitrification preservation The pure cryoprotectant storage tank in the program-controlled cooling device is connected with a constant flow pump, a miniature solenoid valve, and a stainless steel container. The outer wall of the stainless steel container is equipped with a cooling coil, an electric heating wire, and a cooling plate The tube is connected with the refrigeration device, a temperature sensor is installed on the inner wall of the stainless steel container, an electromagnetic stirrer is installed at the bottom of the stainless steel container, and a permanent magnet is installed under the stainless steel container. The permanent magnet is connected with the motor and the speed regulating power supply of the motor. It is connected with a thyristor circuit and a programmable controller, and a constant current pump, a miniature electromagnetic valve, and a temperature sensor are respectively connected with the programmable controller.
所述的不锈钢容器内装有低温保护剂溶液，低温保护剂溶液的用量由恒流泵调节。低温保护剂为二甲亚砜。The cryoprotectant solution is housed in the stainless steel container, and the consumption of the cryoprotectant solution is regulated by a constant flow pump. The cryoprotectant is dimethyl sulfoxide.
本发明具有的有益效果The beneficial effect that the present invention has
本发明通过边降温边提高低温保护剂溶液浓度的方法来尽可能减轻低温保护剂对生物材料的毒性作用，从而提高保存后生物材料的活性。The invention reduces the toxic effect of the cryoprotectant on the biological material as much as possible by increasing the concentration of the cryoprotectant solution while lowering the temperature, thereby improving the activity of the preserved biological material.
本发明通过一个自动控温装置使低温保护剂溶液的温度按照特定的温度曲线下降，并使其温度始终高于溶液冻结点5～10℃，来避免低温保护剂溶液的冻结，保证其均匀扩散。The invention uses an automatic temperature control device to lower the temperature of the cryoprotectant solution according to a specific temperature curve, and keep the temperature always higher than the freezing point of the solution by 5-10°C, so as to avoid freezing of the cryoprotectant solution and ensure its uniform diffusion .
本发明通过可编程控制器、恒流泵、微型电磁阀来控制纯低温保护剂注入低温保护剂溶液存放容器的量，使低温保护剂溶液的浓度按规律速度上升，具有加液均匀、方便、精确的优点。The invention controls the amount of pure cryoprotectant injected into the cryoprotectant solution storage container through a programmable controller, a constant current pump, and a miniature electromagnetic valve, so that the concentration of the cryoprotectant solution rises at a regular speed, and has the advantages of uniform liquid addition, convenience, The advantage of precision.
本发明通过一个电磁搅拌装置对低温保护剂溶液进行搅拌，有利于低温保护剂溶液浓度和温度快速达到均匀。The invention uses an electromagnetic stirring device to stir the cryoprotectant solution, which is beneficial to quickly achieve uniform concentration and temperature of the cryoprotectant solution.
本发明通过把冷却盘管和加热丝缠绕在不锈钢容器外壁，保证了对低温保护剂溶液冷却和加热的均匀性。The invention ensures the uniformity of cooling and heating of the cryoprotectant solution by winding the cooling coil and the heating wire on the outer wall of the stainless steel container.
通过本发明提供的装置，可在20分钟内使得二甲亚砜溶液的浓度由10％提高到49％，同时保持溶液温度始终高于冻结点8℃。Through the device provided by the invention, the concentration of the dimethyl sulfoxide solution can be increased from 10% to 49% within 20 minutes, and at the same time, the temperature of the solution can be kept 8° C. higher than the freezing point.
附图说明 Description of drawings
附图是用于玻璃化保存的低温保护剂溶液程控冷却装置结构示意图，图中：纯低温保护剂储槽1、恒流泵2、微型电磁阀3、可编程控制器4、可控硅电路5、容器盖6、不锈钢容器7、冷却盘管8、电加热丝9、电磁搅拌子10、永磁铁11、电机12、电机调速电源13、制冷装置14、温度传感器15。The accompanying drawing is a schematic diagram of the structure of the cryoprotectant solution program-controlled cooling device for vitrification preservation. In the figure: pure
具体实施方式 Detailed ways
如附图所示，用于玻璃化保存的低温保护剂溶液程控冷却装置中纯低温保护剂储槽1与恒流泵2、微型电磁阀3、不锈钢容器7相连接，不锈钢容器7外壁设有冷却盘管8、电加热丝9，冷却盘管8与制冷装置14相连接，在不锈钢容器7内壁设有温度传感器15，在不锈钢容器7底部设有电磁搅拌子10，在不锈钢容器7下方设有永磁铁11，永磁铁11与电机12、电机调速电源13相连接，电加热丝9与可控硅电路5、可编程控制器4相连接，恒流泵2、微型电磁阀3、温度传感器15分别与可编程控制器4相连接。As shown in the accompanying drawings, the pure
所述的不锈钢容器7内装有低温保护剂溶液，低温保护剂溶液的用量由恒流泵2调节。所述的低温保护剂为二甲亚砜。The cryoprotectant solution is housed in the stainless steel container 7, and the consumption of the cryoprotectant solution is regulated by the
本发明主要由三部分组成：溶液温度控制、溶液浓度控制、溶液搅拌。低温保护剂溶液放置在不锈钢容器中。溶液温度控制部分的作用是使温度按照特定的温度曲线下降，包括制冷装置、冷却盘管、电加热丝、可控硅电路等部件，溶液浓度控制部分的作用使浓度按照规律速度上升，包括恒流泵、纯低温保护剂储槽、微型电磁阀等部件。溶液温度和浓度的控制算法的实施通过可编程控制器。对于溶液温度，通过温度传感器予以检测，传感信号进入可编程控制器转化为数值，与预设温度进行比较，其偏差用PID算法处理，可编程控制器根据处理结果输出电压信号到可控硅电路，控制电加热丝输出的加热量。溶液的浓度通过恒流泵转速大小及开关、微型电磁阀的开关来控制，当恒流泵、微型电磁阀打开时，纯低温保护剂由储槽流向存放溶液的不锈钢容器，溶液的浓度升高，若恒流泵的转速高，则浓度升高的速度快，转速低则升高的速度慢。根据不锈钢容器内初始溶液量、溶液浓度、恒流泵的转速以及恒流泵的开关随时间的变化情况，可以得到不锈钢容器内溶液的浓度随时间的变化情况，进而根据溶液冻结点计算公式得到不锈钢容器内溶液的冻结点随时间的变化情况。在已知当前时间后，可以得到当前溶液的冻结点，溶液的预设温度即根据高于溶液冻结点5～10℃确定。溶液搅拌部分的作用是保证溶液的温度和浓度的均匀性，包括电机、电机调速电源、永磁铁、电磁搅拌子等部件。电机带动永磁铁转动，在电磁力的作用下，电磁搅拌子随永磁铁转动，电机的转动速度可用电机调速电源来调节，转动快则电磁搅拌子的转动也变快，搅拌作用增强。The invention mainly consists of three parts: solution temperature control, solution concentration control and solution stirring. The cryoprotectant solution is placed in a stainless steel container. The function of the solution temperature control part is to make the temperature drop according to a specific temperature curve, including refrigeration devices, cooling coils, electric heating wires, thyristor circuits and other components; the function of the solution concentration control part is to make the concentration rise according to a regular speed, including constant Flow pump, pure cryoprotectant storage tank, miniature solenoid valve and other components. The control algorithm of solution temperature and concentration is implemented by a programmable controller. For the temperature of the solution, it is detected by the temperature sensor, and the sensing signal enters the programmable controller and is converted into a value, which is compared with the preset temperature. The deviation is processed by the PID algorithm, and the programmable controller outputs a voltage signal to the thyristor according to the processing result. The circuit controls the heating output of the electric heating wire. The concentration of the solution is controlled by the speed of the constant-flow pump and the switch of the micro-solenoid valve. When the constant-flow pump and the micro-solenoid valve are turned on, the pure cryoprotectant flows from the storage tank to the stainless steel container storing the solution, and the concentration of the solution increases. , if the speed of the constant flow pump is high, the speed of concentration increase is fast, and if the speed is low, the speed of increase is slow. According to the initial solution volume in the stainless steel container, the solution concentration, the speed of the constant flow pump, and the change of the switch of the constant flow pump with time, the concentration of the solution in the stainless steel container can be obtained. The freezing point of the solution in the stainless steel container changes with time. After the current time is known, the freezing point of the current solution can be obtained, and the preset temperature of the solution is determined based on 5-10°C higher than the freezing point of the solution. The role of the solution stirring part is to ensure the uniformity of the temperature and concentration of the solution, including motors, motor speed control power supplies, permanent magnets, electromagnetic stirrers and other components. The motor drives the permanent magnet to rotate. Under the action of electromagnetic force, the electromagnetic stirrer rotates with the permanent magnet. The rotation speed of the motor can be adjusted by the motor speed regulating power supply. If the rotation is fast, the rotation of the electromagnetic stirrer will also become faster, and the stirring effect will be enhanced.
实施例：Example:
二甲亚砜是一种常见的低温保护剂，常压下玻璃化转变的最低浓度为49％(质量百分比，以下同)。通过本发明提供的装置，可在20分钟内使得二甲亚砜溶液的浓度由10％提高到49％，同时保持溶液温度始终高于冻结点8℃。实施步骤是：1)装置开启前，在不锈钢容器内放10％的二甲亚砜溶液20克，在储槽内放纯二甲亚砜20克；2)开启制冷装置和电加热，使溶液温度下降至10％的二甲亚砜溶液对应的冻结点温度(-4.3℃)以上8℃，即3.7℃，记该时刻为t0；3)于时刻t0打开恒流泵和微型电磁阀，使恒流泵以0.765g/min的速度将纯二甲亚砜泵入不锈钢容器，该泵入过程持续20分钟，使得二甲亚砜溶液的浓度由10％提高到49％，在每一时刻点t，可以计算得到二甲亚砜溶液的浓度为：Dimethyl sulfoxide is a common cryoprotectant, and the minimum concentration of the glass transition under normal pressure is 49% (mass percentage, the same below). Through the device provided by the invention, the concentration of the dimethyl sulfoxide solution can be increased from 10% to 49% within 20 minutes, and at the same time, the temperature of the solution is always kept 8° C. higher than the freezing point. The implementation steps are: 1) before the device is opened, put 20 grams of 10% dimethyl sulfoxide solution in the stainless steel container, and put 20 grams of pure dimethyl sulfoxide in the storage tank; 2) turn on the refrigeration device and electric heating to make the solution The temperature drops to 8°C above the freezing point temperature (-4.3°C) corresponding to the 10% dimethyl sulfoxide solution, that is, 3.7°C, and this time is recorded as t0 ; 3) Open the constant flow pump and the miniature solenoid valve at time t0 , so that the constant flow pump pumps pure dimethyl sulfoxide into the stainless steel container at a speed of 0.765g/min, and the pumping process lasts for 20 minutes, so that the concentration of the dimethyl sulfoxide solution is increased from 10% to 49%. At time point t, the concentration of dimethyl sulfoxide solution can be calculated as:
根据该浓度计算溶液的冻结温度，控制电加热量使得溶液的温度始终比冻结温度高8℃左右。对于实际的生物材料的低温保存，可以在上述过程结束后，关闭电加热，让溶液的温度下降至-80℃以下，再转移至液氮瓶中。在处于玻璃化的状态下，生物材料可以存放很长时间(如3年)而不丧失活性。Calculate the freezing temperature of the solution according to the concentration, and control the electric heating so that the temperature of the solution is always about 8°C higher than the freezing temperature. For the low-temperature storage of actual biological materials, after the above process is completed, turn off the electric heating, let the temperature of the solution drop below -80°C, and then transfer it to a liquid nitrogen bottle. In a vitrified state, biomaterials can be stored for a long time (eg, 3 years) without loss of activity.
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| CN104542575B (en) * | 2015-01-20 | 2016-06-08 | 中国科学技术大学 | Device and method for freezing biological samples |
| CN106234351B (en) * | 2016-07-29 | 2019-06-11 | 浙江大学宁波理工学院 | Device for fluidized loading or removal of cryoprotectants from biological tissue |
| CN107806730A (en) * | 2016-09-09 | 2018-03-16 | 松下电器产业株式会社 | refrigerator |
| CN106839576A (en) * | 2017-02-08 | 2017-06-13 | 上海原能细胞医学技术有限公司 | Refrigerating plant |
| CN109628297B (en) * | 2018-12-14 | 2022-02-18 | 浙江大学宁波理工学院 | Microfluidic high-flux biological sample dripping and freezing storage device |
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| DE10061968A1 (en) * | 2000-12-13 | 2002-06-20 | Friedrich Hoffmann | Vitrification of cornea, employs tissues of rear cornea lamellae including the stroma, Descemet membrane and endothelium |
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