Combined high-flux high-speed cooling and rewarming device

Abstract

A novel combined type high-flux high-speed cooling and rewarming device comprises an upper cover plate, a lower cover plate and one or more sample slides, and when multiple sample slides exist, the sample slides are installed between the upper cover plate and the lower cover plate in a stacked mode; the upper cover plate and the lower cover plate are provided with a sample interface and a sample exhaust port which are respectively communicated with an inner cavity inlet and an inner cavity outlet of the sample slide glass, and the inner cavity inlets and the inner cavity outlets among the slide glass are communicated with each other; a through hole is formed in the center of the sample slide and used for mounting a working medium distributor, and the working medium distributor is arranged among the upper cover plate, the lower cover plate and the sample slide to form multiple layers of height-fixed gaps; the center of the upper cover plate is provided with a cooling and rewarming working medium interface. According to the low-temperature preservation device, a combined structure capable of being conveniently disassembled and assembled is adopted, it is guaranteed that high-speed cooling and rewarming can be conducted on a cooling and rewarming working medium, meanwhile, the flux of the device is rapidly changed, milliliter-level sample cryopreservation can be achieved by stacking the sample slide glass, and the universality and practicability of the low-temperature preservation device are greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of cryopreservation technology, in particular to a combined high-throughput high-speed cooling and reheating device, which is used for ultra-high-speed cooling and reheating of large-throughput biological samples. Background Art

[0002] Cryopreservation is currently the only effective long-term preservation method for many biological materials, and vitrification is an important method in biological cryopreservation. Rapid cooling to avoid the formation of ice crystals inside and outside the cells is a necessary condition for vitrification. In addition, the rewarming rate required for the rewarming process is equivalent to or higher than the critical cooling rate of the cooling process. Insufficient rewarming may lead to cell recrystallization. Therefore, achieving high-speed cooling and rewarming is of great significance for vitrification. However, the current vitrification preservation methods of various biological samples have one or more of the following problems, such as: low cooling and rewarming rate, requiring a higher concentration of cryoprotectant, small freezing flux, difficulty in increasing the freezing flux to the milliliter level or the freezing flux cannot be changed, and specific samples require consumables with specific flux. These problems may lead to the narrow applicability of the vitrification preservation method and make it difficult to apply it to the cryopreservation of various biological samples. Summary of the invention

[0003] The purpose of the present invention is to provide a high-throughput and high-speed cooling and rewarming device that can flexibly change the loading amount. The device provides a new cooling and rewarming device structure that can flexibly change the throughput of sample freezing and provides a convenient and pollution-free biological sample loading method.

[0004] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a combined high-throughput and high-speed cooling and reheating device, comprising an upper cover plate, a lower cover plate and one or more sample slides; the sample slide is a flat hollow structure, and the inner cavity is used to carry the sample; when there are multiple sample slides, they are stacked and installed between the upper cover plate and the lower cover plate, and the inner cavity outlet of each sample slide is sequentially connected to the inner cavity inlet of the sample below it; the upper center of the upper cover plate is provided with a cooling and reheating medium interface and a sample interface, and the lower part of the lower cover plate is provided with a sample exhaust port, the sample interface is connected to the inner cavity inlet of the sample slide below it, and the sample exhaust port is connected to the inner cavity outlet of the upper sample slide, and when the sample is injected from the sample interface, each sample slide is sequentially filled with the sample. The sample slide has a through hole at its center for installing a working fluid distributor. The working fluid distributor has a normal guide ring and a tangential guide structure, and the center of the working fluid distributor is a through structure, wherein the normal guide ring is installed in the central through hole of the sample slide, and the tangential guide structure is located between each sample slide and the upper cover plate and the lower cover plate, separating each sample slide from the upper cover plate and the lower cover plate to form a gap of a specific height. The through structure at the center of each working fluid distributor is aligned and connected with the cooling and reheating working fluid interface of the upper cover plate. When the cooling and reheating working fluid is injected from the cooling and reheating working fluid interface, it will enter each working fluid distributor in turn, and be distributed to the upper and lower surfaces of the sample slide through the tangential guide structure, so as to implement synchronous cooling or reheating of the samples in all sample slides.

[0005] The sample interface of the upper cover plate and the sample exhaust port of the lower cover plate are arranged symmetrically at the outer corners of the upper cover plate and the lower cover plate respectively; the center of the upper cover plate is a through structure, which is connected to the cooling and reheating working medium interface, and an upper positioning groove for positioning the working medium distributor is arranged below the center, and the center of the lower cover plate is a closed structure, and a lower positioning groove for positioning the working medium distributor is arranged above it;

[0006] The sample carrier comprises a first heat exchange plate and a second heat exchange plate which are symmetrically arranged, and a sealed inner frame and a sealed outer frame in the middle. The hollow structure of the sample carrier is formed by the sealed inner frame and the sealed outer frame to form an annular flow channel, and the first heat exchange plate and the second heat exchange plate are tightly combined with the channel to form an annular chamber; the inner cavity inlet is arranged at the corner of the first heat exchange plate, and the inner cavity outlet is correspondingly arranged at the corner of the second heat exchange plate, and the position is centrally symmetrical with the inner cavity inlet;

[0007] The first heat exchanger, the second heat exchanger, the middle sealing inner frame and the sealing outer frame are all made of high thermal conductivity materials, and the outer surfaces of the first heat exchanger and the second heat exchanger are treated with enhanced heat exchange;

[0008] The tangential guide structure of the working fluid distributor is a radial flow channel structure with a positioning boss on the top; the normal guide ring of the working fluid distributor is an annular structure with a groove, the positioning boss is positioned and connected with the upper positioning groove of the upper cover plate or the groove of the normal guide ring of the upper working fluid distributor, and the groove of the normal guide ring is positioned and connected with the lower positioning groove of the lower cover plate or the positioning boss of the upper working fluid distributor.

[0009] The beneficial effects of the present invention are:

[0010] 1) The sample carrier of the present invention can be quickly combined and stacked according to needs to form internal cavity volumes with different fluxes to load biological samples with different fluxes, which has higher flexibility and convenience compared with traditional biological sample freezing methods.

[0011] 2) The present invention can quickly load biological samples through a syringe or the like, and the combined sample slides can be filled step by step through flow channels connected end to end without filling them individually, thereby reducing the risk of contamination during operation and improving the convenience of loading and extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the assembly structure of the present invention;

[0013] Figure 2 It is the explosion schematic diagram of the present invention;

[0014] Figure 3 The structure diagram of the upper cover plate of the present invention is shown in FIG. Figure 1 ;

[0015] Figure 4 The structure diagram of the upper cover plate of the present invention is shown in FIG. Figure 2 ;

[0016] Figure 5 This is a schematic diagram of the structure of the lower cover plate of the present invention. Figure 1 ;

[0017] Figure 6 This is a schematic diagram of the structure of the lower cover plate of the present invention. Figure 2 ;

[0018] Figure 7 is a schematic structural diagram of a sample slide of the present invention;

[0019] Figure 8 yes Figure 7 Explosion diagram of

[0020] Fig. 9 The structure of the working fluid distributor of the present invention is shown in FIG. Figure 1 ;

[0021] Fig.10 The structure of the working fluid distributor of the present invention is shown in FIG. Figure 2 ;

[0022] Fig.11 It is a schematic diagram of the structure of a combination of multiple sample slides of the present invention;

[0023] The markings in the above drawings are: upper cover plate 1, cooling and reheating medium interface 11, sample interface 12, upper positioning groove 13, sample carrier 2, first heat exchange plate 21, second heat exchange plate 22, inner cavity inlet 23, inner cavity outlet 24, sealing inner frame 25, sealing outer frame 26, lower cover plate 3, sample exhaust port 31, lower positioning groove 32, medium distributor 4, tangential guide structure 41, positioning boss 42, normal guide ring 43. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings:

[0025] like Figure 1 , Figure 2 A combined high-throughput high-speed cooling and reheating device is shown, comprising an upper cover plate 1, a lower cover plate 3 and a sample slide 2. The sample slide 2 has a through hole in the center, and is fixedly clamped in the middle by the upper cover plate 1 and the lower cover plate 3. The through hole in the center of the sample slide 2 is inserted into a fixed working fluid distributor 4, and is cross-assembled with the upper cover plate 1, the lower cover plate 3 and the sample slide 2. The working fluid distributor 4 has a normal guide ring 43 and a tangential guide structure 41, and the middle part thereof is a through structure. The tangential guide structure 41 is used to form a specific assembly gap between the upper cover plate 1, the lower cover plate 3 and the sample slide 2. The central part of the upper cover plate 1 has a cooling and reheating working fluid interface 11. The cooling and reheating working fluid enters the working fluid distributor 4 through the cooling and reheating working fluid interface 11, and enters the assembly gaps of each layer through the normal guide ring 43 and the tangential guide structure 41 to perform high-speed heat exchange with the sample slide 2. The corners of the upper cover plate 1 are provided with sample interfaces 12 for injecting biological samples. The lower cover plate 3 has a sample exhaust port 31 at the corner, which is used to exhaust the air in the sample slide 2 when the sample is injected. The sample slide 2 has an inner cavity inlet 23 and an inner cavity outlet 24, which are aligned and connected with the sample interface 12 of the upper cover plate 1 and the sample exhaust port 31 of the lower cover plate 3 respectively.

[0026] Further, such as Figure 3 , Figure 4 As shown, a cooling and reheating medium interface 11 is provided at the center of the upper cover plate 1. The cooling and reheating medium interface 11 is a through hole. An upper positioning groove 13 is provided below the upper cover plate 1. The position of the medium distributor 4 is fixed by the upper positioning groove 13, and the external cooling and reheating medium is introduced through the cooling and reheating medium interface 11 and enters the medium distributor 4 through the through hole. A sample interface 12 is provided at the corner of the upper cover plate 1. The sample interface 12 is sealed with elastic material and can be inserted by a syringe needle to inject biological samples. The lower part of the sample interface 12 corresponds to the inner cavity entrance 23 of the sample slide 2, and the biological sample is injected into the sample slide 2 from this.

[0027] Further, such as Figure 5 , Figure 6 As shown, a lower positioning groove 32 is provided at the center of the upper part of the lower cover plate 3 for positioning the working medium distributor 4. The center of the lower positioning groove 32 is closed, and the cooling and reheating working medium introduced into the working medium distributor 4 can only flow out through the tangential guide structure 41 set by the working medium distributor 4. A sample exhaust port 31 is provided at the corner of the lower cover plate 3. The sample exhaust port 31 is sealed by elastic material, and the upper part thereof corresponds to the inner cavity outlet 24 of the sample slide 2. When injecting the sample, the needle is inserted, and the excess air in the sample slide 2 can be discharged from it.

[0028] Further, such as Figure 7 , Figure 8 The sample carrier 2 shown is a hollow structure, including a first heat exchange plate 21, a second heat exchange plate 22, and a sealed inner frame 25 and a sealed outer frame 26 in the middle. The sealed inner frame 25 and the sealed outer frame 26 form a flow channel. The first heat exchange plate 21 and the second heat exchange plate 22 seal the upper and lower surfaces of the sealed inner frame 25 and the sealed outer frame 26 to form an internal cavity. The sample carrier 2 has a through hole in the center for inserting and assembling the working medium distributor 4; the first heat exchange plate 21 is provided with an inner cavity inlet 23, which is connected to the upper cover plate 1 sample interface 1 2 is aligned and connected, the second heat exchange plate 22 is provided with an inner cavity outlet 24, which is aligned and connected with the sample exhaust port 31 of the lower cover plate 3, and the inner cavity inlet 23 and the inner cavity outlet 24 are arranged symmetrically with each other. When injecting a sample, it enters from the inner cavity inlet 23, gradually fills the hollow structure and discharges the excess gas from the aligned inner cavity outlet 24. When a multi-stage sample carrier 2 is set, the inner cavity outlet 24 of the upper stage sample carrier 2 is connected with the inner cavity inlet 23 of the lower stage sample carrier 2, so that the step-by-step filling of each stage sample carrier 2 can be realized. The outer surfaces of the first heat exchange plate 21 and the second heat exchange plate 22 can be subjected to enhanced heat exchange treatment to enhance the cooling and reheating effect.

[0029] Further, such as Fig. 9 , Fig.10 The working fluid distributor 4 shown has a normal guide ring 43 and a tangential guide structure 41. The tangential guide structure 41 is a radial flow channel structure and has a positioning boss 42 on the top, which is positioned and linked to the upper positioning groove 13 of the upper cover plate 1. The normal guide ring 43 has a groove structure, which is positioned and linked to the lower positioning groove 32 of the lower cover plate 3. The installation of the working fluid distributors 4 is connected and positioned by the positioning boss 42 and the groove structure of the normal guide ring 43. The stacked working fluid distributors 4 form a specific gap between the upper cover plate 1, the lower cover plate 3 and the sample carrier 2 through the tangential guide structure 41. The cooling and reheating working fluid introduced by the cooling and reheating working fluid interface 11 of the upper cover plate 1 is introduced through the normal guide ring 43 and radially flows out of the gap formed by the support of the tangential guide structure 41 to perform high-speed heat exchange with the surface of each sample carrier 2.

[0030] Further, such as Fig.11As shown, when combining multiple sample slides 2, it is only necessary to correspond the inner cavity outlet 24 and the inner cavity inlet 23 of the sample slide 2 and add a centrally mounted working fluid distributor 4.

[0031] The working principle of the present invention is as follows:

[0032] (1) Combined variable high-throughput structure. The specially designed sample slides and cross-mounted fluid distributors can quickly change the overall structure flux, greatly increasing the applicability and flexibility. By stacking sample slides, milliliter-level sample freezing can be achieved.

[0033] (2) Continuous filling without pollution. The sample carrier and the housing are connected by a continuous end-to-end flow channel to achieve the loading of each level of the sample carrier, without the need for a single filling combination, reducing operational risks.

[0034] The cooling and reheating process of the present invention can be controlled and operated based on a working fluid drive and control system. The specific structure of the working fluid drive and control system can be found in the prior art and will not be described in detail here.

[0035] The cooling and reheating process of the present invention is as follows:

[0036] First, prepare the cell fluid to be tested and a sterile syringe, and use the sterile syringe to extract the required amount of cell fluid for later use.

[0037] Second, insert the syringe loaded with cell fluid into the sample interface 12 of the upper cover plate 1, and insert the needle of a syringe or an infusion tube into the sample exhaust port 31 of the lower cover plate 3 to facilitate exhausting air during injection.

[0038] Third, gently push in the required amount of cell solution, and stop when liquid is observed to appear in the sample exhaust port 31 of the lower cover plate 3 , then remove the needle from the sample exhaust port 31 , and pull out the syringe from the sample interface 12 of the upper cover plate 1 .

[0039] Fourth, prepare the working fluid drive control system and liquid nitrogen required for the experiment, connect the working fluid interface 11 of the combined high-throughput and high-speed cooling and reheating device with the end of the pipeline of the liquid nitrogen jet of the working fluid drive control system, and then arrange an insulation box at the bottom to collect the liquid nitrogen of the jet.

[0040] Fifth, open the pipeline of the working fluid drive and control system to complete the jet impact cooling process, then separate the liquid nitrogen tank line and the cooling and recooling working fluid interface 11 of the combined high-throughput and high-speed cooling and recooling device, remove it from the pipeline and drop it into the liquid nitrogen in the bottom insulation box, thus completing the cooling experiment process.

[0041] Sixth, the combined high-throughput and high-speed cooling and reheating device in the insulation box is adjusted to align and connect with the reheating pipeline of the working fluid drive control system using tweezers, and the box body is used for temporary insulation.

[0042] Seventh, prepare the working fluid drive control system and rewarming working fluid required for the experiment, then open the pipeline of the working fluid drive and control system to complete the jet impact rewarming process, and then separate the rewarming pipeline and the rewarming working fluid interface 11 of the combined high-throughput and high-speed rewarming device, so that it falls into the insulation box loaded with the rewarming working fluid for insulation.

[0043] Eighth, take out the combined high-throughput and high-speed cooling and reheating device from the heat preservation box, wipe off the moisture on its surface, prepare a sterile syringe and needle, insert the sterile syringe into the sample interface 12 on the upper cover plate 1, and insert the needle into the sample exhaust port 31 of the lower cover plate 3.

[0044] Ninth, pull the sterile syringe to slowly extract the sample in the combined high-throughput high-speed cooling and rewarming device until all the extractions are completed, pull out the sterile syringe, remove the needle from the sample exhaust port 31, transfer the cell fluid in the syringe, and complete the cell rewarming experiment process.

[0045] Embodiment 1:

[0046] In this embodiment, the material of the upper cover plate 1 and the lower cover plate 3 is PMMA, and the overall size is 45*45*5mm; the inner diameter of the cooling and reheating medium interface 11 of the upper cover plate 1 is 5mm; the diameter of the upper positioning groove is 8mm, and the depth is 0.3mm; the diameter of the lower positioning groove of the lower cover plate is 9.1mm, and the depth is 0.6mm; the first and second heat exchange plates 22 of the sample carrier 2 are made of aluminum nitride ceramic sheets, and the size is 35*35*0.2mm, and the enhanced heat exchange treatment on the surface is a fork-shaped bionic microchannel, and the channel depth is 0.1mm; the sealed inner frame 25 and the sealed outer frame 26 in the middle of the sample carrier 2 are also made of aluminum nitride ceramic sheets, The outer diameter of the hollow structure is 29mm, the inner diameter is 15mm, and the thickness is 0.3mm. The overall size of the sample slide is 35*35*0.6mm, which can carry about 0.15ml of cell fluid; the diameter of the through hole in the middle of the sample slide 2 is 9.1mm; the working fluid distributor 4 is arranged in two layers, the flow channel height of the tangential guide structure 41 is 0.8mm, and its maximum diameter is 15mm, the positioning boss 42 is 0.3mm high and has a diameter of 7.8mm, the outer diameter of the normal guide ring 43 is 9mm, the height is 0.6mm, the groove diameter is 8mm, the groove depth is 0.3mm, and the diameter of the center through hole is 5mm.

[0047] According to the above configuration, this embodiment is equipped with a sample carrier 2, and the interlayer height of the working fluid flow on both sides of the sample carrier 2 is 0.8mm, and a total of 0.15ml of cell fluid can be loaded for cooling and reheating test. According to the test results, the cooling rate of this embodiment can reach an average of 190℃ / s, and the reheating rate can reach 960℃ / s.

[0048] Embodiment 2:

[0049] like Fig.11 As shown, in order to increase the biological sample flux used for each cooling and reheating, the sample slides 2 of the present invention are combined into three pieces; the corresponding number of working fluid distributors 4 is increased to four, and a working fluid distributor 4 is arranged between each of the upper cover plate 1, the lower cover plate 3 and the sample slides 2, and the sample interfaces 12, the inner cavity inlet 23, the inner cavity outlet 24 and the sample exhaust port 31 between each level are installed one by one. After the flux is increased, the cooling and reheating working fluid will be introduced into the surface of all sample slides 2 by the working fluid distributor 4 to achieve a high-speed cooling and reheating effect.

[0050] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A combined high-throughput high-speed cooling and reheating device, characterized in that: The invention comprises an upper cover plate (1), a lower cover plate (3) and one or more sample slides (2); the sample slides (2) are flat and hollow in structure, and the inner cavity is used to carry samples; when there are multiple sample slides (2), they are stacked and installed between the upper cover plate (1) and the lower cover plate (3), and the inner cavity outlet (24) of each sample slide (2) is sequentially connected to the sample inner cavity inlet (23) below it; the upper cover plate (1) has a sample interface (12) on the top, and the lower cover plate (3) has a sample exhaust port (31) on the bottom; the sample interface (12) is connected to the inner cavity inlet (23) of the sample slide (2) below it, and the sample exhaust port (31) is connected to the inner cavity outlet (24) of the upper sample slide (2) to form a continuous sample passage, and when the sample is injected from the sample interface (12), each sample slide (2) is sequentially filled; the sample slide (2) has a through hole in the center for installing a working medium distributor; The working fluid distributor (4) comprises a normal flow guide ring (43) and a tangential flow guide structure (41), wherein the normal flow guide ring (43) is installed in the central through hole of the sample slide, and the tangential flow guide structure (41) is located between each sample slide (2) and the upper cover plate (1) and the lower cover plate (3), and separates each sample slide (2) from the upper cover plate (1) and the lower cover plate (3) to form a gap of a specific height; the upper cover plate (1) has a cooling and reheating working fluid interface (11) at the center, which is aligned and connected with the normal flow guide ring (43) of each working fluid distributor (4); when the cooling and reheating working fluid is injected from the cooling and reheating working fluid interface (11), it will enter each working fluid distributor (4) in turn, and be distributed to the upper and lower surfaces of the sample slide (2) through the tangential flow guide structure (41), and the samples in all the sample slides (2) are subjected to synchronous high temperature cooling or reheating through high-speed shear flow on the surface of the sample slide (2).

2. The combined high-throughput high-speed cooling and reheating device according to claim 1, characterized in that: The sample interface (12) of the upper cover plate (1) and the sample exhaust port (31) of the lower cover plate (3) are arranged symmetrically at the outer corners of the upper cover plate (1) and the lower cover plate (3); the center of the upper cover plate (1) is a through structure, which is connected to the cooling and reheating working medium interface (11), and an upper positioning groove (13) for positioning the working medium distributor (4) is arranged below the center; the center of the lower cover plate (3) is a closed structure, and a lower positioning groove (32) for positioning the working medium distributor (4) is arranged above it.

3. The combined high-throughput high-speed cooling and reheating device according to claim 1, characterized in that: The sample carrier (2) comprises a first heat exchange plate (21) and a second heat exchange plate (22) which are symmetrically arranged, and a sealed inner frame (25) and a sealed outer frame (26) in the middle; the hollow structure of the sample carrier is formed by the sealed inner frame (25) and the sealed outer frame (26) to form an annular flow channel, and the first heat exchange plate (21) and the second heat exchange plate (22) are tightly combined with it to form an annular chamber; the inner cavity inlet (23) is arranged at the corner of the first heat exchange plate (21), and the inner cavity outlet (24) is correspondingly arranged at the corner of the second heat exchange plate (22), and the position is centrally symmetrical with the inner cavity inlet (23).

4. The combined high-throughput high-speed cooling and reheating device according to claim 3 is characterized in that: The first heat exchange plate (21), the second heat exchange plate (22), the intermediate sealing inner frame (25) and the sealing outer frame (26) are all made of high thermal conductivity materials, and the outer surfaces of the first heat exchange plate (21) and the second heat exchange plate (22) are both subjected to enhanced heat exchange treatment.

5. The combined high-throughput high-speed cooling and reheating device according to claim 1, characterized in that: The tangential flow guide structure (41) of the working fluid distributor (4) is a radial flow channel structure, and has a positioning boss (42) on the top; the normal flow guide ring (43) of the working fluid distributor (4) is an annular structure with a groove, the positioning boss (42) is connected to the upper positioning groove (13) of the upper cover plate (1) or the groove of the normal flow guide ring (43) of the upper working fluid distributor (4) in a positioning manner, and the groove of the normal flow guide ring (43) is connected to the lower positioning groove (32) of the lower cover plate (3) or the positioning boss (42) of the upper working fluid distributor (4) in a positioning manner.

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