An upper chip and a microdevice with multi-stage addition of cryoprotectant for cells

Abstract

The present invention discloses an upper chip and a micro-device with multi-stage addition of cryoprotectant. The upper chip is provided with a cryoprotectant addition liquid generation area and a cryoprotectant stepwise addition area. The cryoprotectant addition liquid generation area includes a first liquid inlet channel and a first processing unit, and the first processing unit includes a plurality of first mixers connected in series; the cryoprotectant stepwise addition area includes a plurality of second processing units connected in series; each first mixer and each second processing unit are arranged in reverse order and paired. The first processing unit is provided with a multi-stage first mixer for diluting to obtain a cryoprotectant addition liquid with a gradient concentration, and then flowing the cryoprotectant addition liquid into the cryoprotectant stepwise addition area in stages to be mixed and separated and concentrated with the cell suspension in stages, adding the cryoprotectant step by step, reducing the mechanical damage and osmotic pressure damage of cells, avoiding cell dilution, improving the addition efficiency, and reducing the cost. The present invention can be widely applied to the technical field of medical devices.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and particularly relates to an upper-layer chip and a micro-device for multi-stage addition of cryoprotectant to cells. Background Art

[0002] Cryopreservation is an important technology for the preservation of precious cells in the fields of reproductive medicine, tissue regeneration, and cell therapy. Cryoprotectants (such as dimethyl sulfoxide DMSO, glycerol, propylene glycol, ethylene glycol, etc.) can prevent cells from suffering from cryo-injury during cryopreservation and improve the survival ability of cells after thawing. Therefore, cryoprotectants need to be added before cell cryopreservation. However, during the addition of cryoprotectants, due to the imbalance of osmotic pressure inside and outside the cells, the cells will suffer from severe osmotic injury. Therefore, it is extremely important to safely and effectively load cryoprotectants into cell samples.

[0003] Traditionally, the stepwise loading method is often used to load cryoprotectants into cell samples. For example, ethylene glycol is stepwise loaded into human oocytes, and cryoprotectants are gradually added to neural stem cells by using an isosmotic pressure difference and an osmotic time interval. Although the distributed addition method can reduce cell osmotic injury, the operation procedure is cumbersome, time-consuming and laborious. During the repeated operation process, multiple mechanical stresses and repeated cell transfers are likely to cause mechanical damage and cell loss to the cells. At the same time, the open environment will cause cell contamination.

[0004] Microfluidic technology is expected to control the behavior of cells and the solution environment in which the cells are located during the addition of cryoprotectants and realize the automation of multi-step addition due to the small amount of sample required, automation, and the ability to precisely control cells and the osmotic pressure environment in which the cells are located. Furthermore, it can reduce cell permeation injury during the addition of cryoprotectants, simplify the operation process, and reduce the possibility of cell loss and contamination. Currently, there are already microfluidic devices based on the principle of laminar interface diffusion and the principle of micro-membrane dialysis to achieve the addition of cryoprotectants for precious trace cells, but the addition efficiency is low, and the cells are easily lost and diluted. Summary of the Invention

[0005] In order to solve at least one of the above technical problems and reduce the mechanical injury and osmotic injury of cells, the present invention provides an upper-layer chip and a micro-device for multi-stage addition of cryoprotectant to cells, and the technical solutions adopted are as follows:

[0006] The upper chip of the multi-stage cryoprotectant adding cell cryoprotectant provided by the present invention is provided with a cryoprotectant adding liquid generation area and a cryoprotectant stepwise adding area. The cryoprotectant adding liquid generation area includes a first liquid inlet channel and a first processing unit. The first processing unit includes a plurality of first mixers arranged in series. The first liquid inlet channel is used to introduce a diluent into each of the first mixers. Each of the first mixers is provided with a first liquid outlet channel. The cryoprotectant stepwise adding area includes a plurality of second processing units arranged in series. The number of the second processing units is the same as the number of the first mixers. Each of the second processing units is provided with a second liquid inlet and a second liquid outlet. The second liquid inlet is used to communicate with the first liquid outlet channel. Each of the second processing units includes a second mixer and a cell separation and concentration module arranged in series. The second liquid inlet is arranged at the inlet of the second mixer. The second liquid outlet is arranged at the outlet of the cell separation and concentration module. Among them, each of the first mixers in the cryoprotectant adding liquid generation area is arranged in reverse order pairing with each of the second processing units in the cryoprotectant stepwise adding area. The first liquid outlet channel of the first mixer ranked first in the cryoprotectant adding liquid generation area is used to communicate with the second liquid inlet of the second processing unit ranked last in the cryoprotectant stepwise adding area. The first liquid outlet channel of the first mixer ranked last in the cryoprotectant adding liquid generation area is used to communicate with the second liquid inlet of the second processing unit ranked first in the cryoprotectant stepwise adding area.

[0007] In some embodiments of the present invention, the upper chip is provided with a third inlet, and the third inlet is used to introduce a cell suspension into the cryoprotectant stepwise adding area. The third inlet is used to communicate with the second liquid inlet of the second processing unit ranked first in the cryoprotectant stepwise adding area.

[0008] In some embodiments of the present invention, the upper chip is provided with a second inlet, and the second inlet is used to introduce a cryoprotectant stock solution into the first mixer ranked first in the first processing unit.

[0009] In some embodiments of the present invention, the upper chip is provided with a waste liquid collection area. Each of the second processing units is provided with a second waste liquid discharge channel for communicating with the waste liquid collection area. The second waste liquid discharge channel is arranged at the outlet of the cell separation and concentration module.

[0010] In some embodiments of the present invention, the waste liquid collection area is provided with a plurality of curved paths for extending the channel length.

[0011] In some embodiments of the present invention, the first liquid inlet channel is provided with a plurality of curved paths for extending the channel length.

[0012] In some embodiments of the present invention, the first liquid inlet channel is provided with a plurality of first flow channels, the number of the first flow channels is the same as that of the first mixers, each of the first flow channels is arranged in a paired manner with each of the first mixers, and the channel section of the first liquid inlet channel between two adjacent first flow channels is provided with the bending path.

[0013] In some embodiments of the present invention, each of the first liquid outlet channels is provided with a bending path for extending the channel length.

[0014] In some embodiments of the present invention, the upper chip is provided with a fourth outlet for discharging the cell suspension, and the fourth outlet is used to communicate with the second liquid outlet of the second processing unit arranged at the end in the cryoprotectant stepwise addition area.

[0015] The micro-device for multi-stage addition of cell cryoprotectant provided by the present invention includes a lower chip and an upper chip.

[0016] The embodiments of the present invention have at least the following beneficial effects: The first processing unit in the cryoprotectant addition liquid generation area is provided with a multi-stage first mixer for diluting to obtain a cryoprotectant addition liquid with a gradient concentration, and then the cryoprotectant addition liquid is fed into the cryoprotectant stepwise addition area in a graded manner to be mixed and separated and concentrated with the cell suspension in a graded manner, so as to achieve the addition of the cryoprotectant step by step, add the cryoprotectant more safely and effectively, reduce the mechanical damage and osmotic pressure damage of cells, avoid cell dilution, and improve the cryoprotectant addition efficiency. The present invention can be widely applied to the technical field of medical devices. Description of the Drawings

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein:

[0018] Figure 1 is the structural diagram of the upper chip;

[0019] Figure 2 is the design schematic diagram of the upper chip. Detailed Description of the Embodiments

[0020] The following will be combined with Figures 1 to 2 Describe the embodiments of the present invention in detail. The examples of the embodiments are shown in the drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0021] In the description of the present invention, it should be understood that if terms such as "center", "middle part", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, it is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. The features defined with "first" and "second" are used to distinguish the feature names and do not have special meanings. In addition, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0022] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "mounted", "connected" and "coupled" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0023] The present invention relates to an upper chip for adding a cryoprotectant to cells in multiple stages. The upper chip is provided with a cryoprotectant addition liquid generation area 6 and a cryoprotectant stepwise addition area 8. The cryoprotectant addition liquid generation area 6 is used to generate a variety of cryoprotectant addition liquids with the same or different flow rates and gradient concentrations. The cryoprotectant stepwise addition area 8 is used to mix and separate and concentrate the cryoprotectant addition liquid with the cell suspension step by step in the order of increasing concentration to obtain the treated cell suspension, so as to safely and effectively add the cryoprotectant to the cells.

[0024] The cryoprotectant additive solution generation area 6 includes a first liquid inlet channel and a first processing unit. The first processing unit includes a plurality of first mixers arranged in series. The first mixers are set as Tesla mixers to achieve rapid mixing. The first liquid inlet channel is used to introduce the diluent into the first processing unit. The diluent is DMEM medium containing 40% fetal bovine serum. Specifically, the first liquid inlet channel is used to introduce the diluent into each first mixer. Further, the upper chip is provided with a first inlet 1, and the first inlet 1 is set as a through hole penetrating the upper chip. The first inlet 1 is used to introduce the diluent into the first liquid inlet channel. Further, the upper chip is provided with a second inlet 2, and the second inlet 2 is set as a through hole penetrating the upper chip. The second inlet 2 is used to introduce the original cryoprotectant solution into the first processing unit. Specifically, the second inlet 2 is used to introduce the original cryoprotectant solution into the first mixer ranked first in the first processing unit. It can be understood that the diluent gradually converges with each first mixer in the first processing unit. According to the series order, the concentration of the cryoprotectant additive solution discharged from the later first mixer is lower.

[0025] Further, the first liquid inlet channel is provided with a plurality of bending paths for extending the channel length, and the bending paths are set as S-shaped or annular. Combining with the attached drawings, the first liquid inlet channel is provided with a plurality of first flow channels, and the number of the first flow channels is the same as that of the first mixers. Each first flow channel is paired with each first mixer. The channel section of the first liquid inlet channel between two adjacent first flow channels is provided with a bending path, and the length of the bending path is used to control the flow rate of the liquid in the micro-device.

[0026] Combining with the attached drawings, each first mixer is provided with a first liquid outlet channel, and each first liquid outlet channel is used to introduce the cryoprotectant additive solution into the cryoprotectant stepwise addition area 8. Further, the upper chip is provided with a third inlet 3, and the third inlet 3 is set as a through hole penetrating the upper chip. The third inlet 3 is used to introduce the cell suspension into the cryoprotectant stepwise addition area 8. The cell suspension is mixed, separated and concentrated with the cryoprotectant additive solution with a gradient concentration added step by step in the cryoprotectant stepwise addition area 8. Specifically, the upper chip is provided with a cell suspension addition area 7, the third inlet 3 is connected to the cell suspension addition area 7, and the cell suspension addition area 7 is connected to the cryoprotectant stepwise addition area 8.

[0027] The cryoprotectant stepwise addition area 8 includes a plurality of second processing units arranged in series. The second processing units are used to mix, separate and concentrate the cell suspension and the cryoprotectant addition solution. Specifically, the number of the second processing units is the same as that of the first mixers, and the second processing units are paired with the first mixers to achieve stepwise and gradual addition of the cell cryoprotectant addition solution. Further, each second processing unit is provided with a second liquid inlet and a second liquid outlet. The second liquid inlet is used to connect to the first liquid outlet channel, and the third inlet 3 is used to connect to the second liquid inlet of the second processing unit ranked first in the cryoprotectant stepwise addition area 8.

[0028] Further, each first mixer in the cryoprotectant addition solution generation area 6 is paired with each second processing unit in the cryoprotectant stepwise addition area 8 in reverse order. Specifically, the first liquid outlet channel of the first mixer ranked first in the cryoprotectant addition solution generation area 6 is used to connect to the second liquid inlet of the second processing unit ranked last in the cryoprotectant stepwise addition area 8, and the first liquid outlet channel of the first mixer ranked last in the cryoprotectant addition solution generation area 6 is used to connect to the second liquid inlet of the second processing unit ranked first in the cryoprotectant stepwise addition area 8. It can be understood that in the cryoprotectant stepwise addition area 8, in the series order, the concentration of the cryoprotectant addition solution introduced into the later second processing unit is higher. The cell suspension introduced through the third inlet 3 flows step by step in the cryoprotectant stepwise addition area 8, first mixes and reacts with the cryoprotectant addition solution with a low concentration, and the separated and concentrated cell suspension enters the next second processing unit to continue mixing, separating and concentrating.

[0029] Compared with the traditional time-consuming stepwise addition method and the microfluidic devices based on laminar diffusion and membrane dialysis, adopting this method of hierarchical mixing, separation and concentration can add the cryoprotectant more safely and effectively, reduce the mechanical damage and osmotic pressure damage of cells, ensure that the cell volume changes within a safe range, avoid cell dilution, improve the cryoprotectant addition efficiency, and save storage capacity and cost.

[0030] Each second processing unit includes a second mixer and a cell separation and concentration module arranged in series. The second mixer is set as a Tesla mixer to achieve rapid mixing. The second liquid inlet is arranged at the inlet of the second mixer, and the second liquid outlet is arranged at the outlet of the cell separation and concentration module. In two adjacent second processing units, in the series order, the second liquid outlet of the cell separation and concentration module in the previous second processing unit is connected to the second mixer in the next second processing unit. Further, the upper chip is provided with a fourth outlet 4 for discharging the cell suspension. The fourth outlet 4 is set as a through hole penetrating the upper chip, and the fourth outlet 4 is used to connect to the second liquid outlet of the second processing unit ranked last in the cryoprotectant stepwise addition area 8. Specifically, the cell suspension processed step by step in the cryoprotectant stepwise addition area 8 is discharged from the fourth outlet 4.

[0031] The upper chip is provided with a waste liquid collection area 9 for recycling the waste liquid. Each second processing unit is provided with a second waste liquid discharge channel for communicating with the waste liquid collection area 9. Specifically, the second waste liquid discharge channel is arranged at the outlet of the cell separation and concentration module. After the cell separation and concentration module separates and concentrates the cells, a cell suspension and cell-free waste liquid are obtained. With reference to the accompanying drawings, the upper chip is provided with a fifth outlet 5 for discharging the waste liquid. The fifth outlet 5 is arranged as a through hole penetrating the upper chip, and the fifth outlet 5 communicates with the waste liquid collection area 9. Further, the waste liquid collection area 9 is provided with a plurality of bending paths for extending the channel length. The bending paths are arranged in an S shape or a ring shape, and the length of the bending paths is used to control the flow rate of the liquid in the micro-device.

[0032] With reference to the accompanying drawings, each first liquid outlet channel is provided with a bending path for extending the channel length. The bending paths are arranged in an S shape or a ring shape, and the length of the bending paths is used to control the flow rate of the liquid in the micro-device.

[0033] It can be understood that the cryoprotectant additive solution generation area 6, the cryoprotectant stepwise addition area 8, and the waste liquid collection area 9 are arranged on the same side of the upper chip. Further, the cryoprotectant additive solution generation area 6, the cryoprotectant stepwise addition area 8, and the waste liquid collection area 9 are arranged as recessed structures on the surface of the upper chip.

[0034] The present invention relates to a micro-device for multi-stage addition of cryoprotectant to cells. The micro-device includes a lower chip and an upper chip. The upper chip is arranged on the lower chip. The cryoprotectant additive solution generation area 6 and the cryoprotectant stepwise addition area 8 are arranged on the side of the upper chip for attaching to the lower chip.

[0035] The upper chip is arranged in a square or circular shape, and the material is made of organic glass or polydimethylsiloxane; the lower chip is arranged in a square or circular shape, and the material is made of organic glass or polydimethylsiloxane. The following will introduce the steps for the micro-device to achieve multi-stage addition of cryoprotectant to cells, mainly including: generation of cryoprotectant additive solution with gradient concentration; stepwise addition of cryoprotectant; waste liquid collection.

[0036] The cryoprotectant stock solution is introduced into the cryoprotectant additive solution generation area 6 through the second inlet 2, and the diluent is introduced into the first liquid inlet channel through the first inlet 1. After being quickly mixed by the first mixer, it is divided into two parts. One part of the cryoprotectant additive solution flows into the second mixer of the second processing unit through the first liquid outlet channel, and the other part of the cryoprotectant additive solution flows into the next-stage first mixer to continue dilution to obtain a lower concentration, thereby generating a cryoprotectant additive solution with gradient concentration.

[0037] The cell suspension is introduced into the cryoprotectant stepwise addition area 8 through the third inlet 3. After the cell suspension and the cryoprotectant addition solution are rapidly mixed in the second mixer, the addition is completed, and it enters the cell separation and concentration module. In the cell separation and concentration module, the cells in the cell suspension gather towards one side of the channel under the action of the lift induced by shear in the channel, the centripetal lift induced by the high pressure generated between the wall and the particles, and the DEAN drag force of the DEAN vortex. The cell separation and concentration module divides the solution into a cell suspension containing cells and a waste liquid without cells. The cell suspension containing cells enters the next-stage second treatment unit, thus completing the addition of cryoprotectant with gradient concentration and cell separation and concentration.

[0038] The schematic diagram of the upper-layer chip channel is as Figure 2 shown. The flow rate and flow resistance satisfy the microfluidic equivalent Kirchhoff's current and voltage laws, specifically:

[0039]

[0040] q m，1 = q c，1 × c hype / c d，1

[0041] q c，i+1 = q m，i - q d，i (i = 1, 2, 3)

[0042] q m，i+1 = q c，i+1 × c d，i / c d，i+1 (i = 1, 2, 3)

[0043] q b，i = q m，i - q c，i (i = 1, 2, 3, 4)

[0044] q a，i = q b，i + q a，i+1 (i = 1, 2, 3)

[0045] q e，i = q d，i + q n，i+1 (i = 1, 2, 3, 4)

[0046] q n，i = q e，i - q f，i (i = 1, 2, 3, 4)

[0047] q g，i = q f，i + q g，i+1 (i = 1, 2, 3)

[0048] q a，i+1 ×r a，i+1 +q b，i+1 ×r b，i+1 =q b，i ×r b，i +q m，i ×r m，i +q c，i+1 ×r c，i+1 (i = 1, 2, 3)

[0049] q c，i+1 ×r c，i+1 +q m，i+1 ×r m，i+1 +q d，i+1 ×r d，i+1 +q e，i+1 ×r e，i+1 +q n，i+1 ×r n，i+1 =q d，i ×r d，i (i = 1, 2, 3)

[0050] q n，i+1 ×r n，i+1 +q e，i ×r e，i +q f，i ×r f，i =q f，i +1×r f，i+1 +q g，i+1 ×r j，i+1 (i = 1, 2, 3)

[0051] Wherein, q d，i =q f，i =q n，5 =0.1 mL / min (i = 1, 2, 3, 4); c hype =100%, c d，1 =12.5%, c d，2 =10%, c d，3 =7.5%, c d，4 =0.5%; r b，i =2.4 mm, r m，i =15.4 mm, r e，i =24.1 mm, r f，i =2.94 mm, r n，i =4 mm (i = 1, 2, 3, 4); r c，i =0.6 mm (i = 2, 3, 4); r c，1 =r a，1 =r g，1 =r n，5 =2 mm; r d，4= 6 mm; q a，4 = q b，4 , q g，4 = q f，4 。

[0052] Where q is the flow rate, r is the channel flow resistance (equivalent to length), a represents the channel through which the diluent flows, b represents the channel through which the diluent flows to each stage of the cryoprotectant solution, c represents the channel through which the cryoprotectant solution flows to the next stage, m represents the channel where the diluent and the cryoprotectant solution are mixed, d represents the channel through which the cryoprotectant additive solution flows to each stage of the cell solution, n represents the channel through which the cell solution flows to the next stage, e represents the channel of the cryoprotectant stepwise addition area, f represents the channel for collecting waste liquid at each stage, g represents the channel for collecting the waste liquid after the confluence of the waste liquids at each stage, and i represents the number of stages.

[0053] The following lists two specific embodiments to supplement the description of the microdevice.

[0054] Embodiment 1

[0055] Four first mixers are provided in the first processing unit in the cryoprotectant additive solution generation area 6. The diluent and the cryoprotectant stock solution are combined in the channel of the cryoprotectant additive solution generation area 6 at a certain flow rate ratio and uniformly mixed in the first mixer. After that, a part of the generated diluted cryoprotectant solution enters the cryoprotectant stepwise addition area 8 to perform the addition of the cryoprotectant, and another part of the diluted cryoprotectant solution is mixed with the diluent again at a certain flow rate ratio in the next-stage first mixer, successively realizing the generation of cryoprotectant additive solutions with four gradient concentrations of 12.5%, 10%, 7.5%, and 5% DMSO and a flow rate of 0.1 mL / min for each.

[0056] Four second mixers and a cell separation and concentration module are provided in the cryoprotectant stepwise addition area 8. The 0.1 mL / min diluted cryoprotectant solution and the 0.1 mL / min cell suspension are combined in the channel of the cryoprotectant stepwise addition area 8 at a 1:1 flow rate ratio and uniformly mixed in the second mixer. After the addition of the cryoprotectant to the cells at this stage is completed, the cryoprotectant-added cell suspension enters the cell separation and concentration module to perform cell separation and concentration. The cell-free solution enters the waste liquid collection area 9 at a flow rate of 0.1 mL / min and is discharged through the fifth outlet 5. The cell-containing solution at a flow rate of 0.1 mL / min is mixed with the diluted cryoprotectant solution of another concentration at a flow rate ratio of 1:1 for the next stage, successively realizing the addition of four gradient concentrations of cryoprotectant (12.5%, 10%, 7.5%, 5% DMSO) and cell separation and concentration.

[0057] Solutions with DMSO volume fractions of 12.5%, 10%, 7.5%, and 5% and all with a flow rate of 0.1 mL / min are used as cryoprotectant addition solutions at four gradient concentrations. After being respectively combined and mixed with a cell suspension at 0.1 mL / min and entering, through rapid mixing, separation, and concentration, the cryoprotectant can be added more safely, effectively, and automatically, reducing mechanical damage and osmotic damage to cells, avoiding cell dilution, improving the cryoprotectant addition efficiency, and saving storage capacity and costs.

[0058] Example Two

[0059] Fetal bovine serum is added to DMEM medium and mixed evenly to obtain a dilution containing 40% (mass / volume ratio) fetal bovine serum. Cultured cells MCF-7 are processed to obtain a cell suspension of 1×10 6 cells / mL. The dilution, cryoprotectant stock solution (100% DMSO), and cell suspension are respectively introduced into the micro-device from the first inlet 1, the second inlet 2, and the third inlet 3 at flow rates of 0.365 mL / min, 0.035 mL / min, and 0.1 mL / min.

[0060] The dilution and the cryoprotectant stock solution flow along the channels in the cryoprotectant addition solution generation area 6 and are evenly mixed when reaching the mixer, gradually forming cryoprotectant addition solutions with 12.5%, 10%, 7.5%, and 5% DMSO and respectively flowing into the cryoprotectant stepwise addition area 8 at a flow rate of 0.1 mL / min. Subsequently, the cell suspension at 0.1 mL / min successively converges with the cryoprotectant addition solutions of 5%, 7.5%, 10%, and 12.5% DMSO for cells in the cryoprotectant stepwise addition area 8 at a flow ratio of 1:1. The 0.2 mL / min combined liquid is rapidly and evenly mixed through mixing to achieve the addition of the cryoprotectant, and the diluted cells are concentrated through the cell separation and concentration module. The 0.2 mL / min cell suspension is concentrated into a 0.1 mL / min cell suspension, and the addition of the cryoprotectant and the concentration of the diluted cell suspension after addition are realized step by step. The cryoprotectant addition solutions for cells with multi-stage gradient concentrations can reduce the osmotic damage to cells and ensure that the cell volume changes within a safe range; at the same time, the multi-stage stepwise concentration of the cell suspension avoids cell dilution, improves the cryoprotectant addition efficiency, and saves storage capacity and costs.

[0061] The cell suspension flowing to the fourth outlet 4 and the concentrated waste liquid flowing to the fifth outlet 5 flow out of the micro-device through a medical rubber hose. After the micro-device operates stably, samples are taken at the fourth outlet 4 and the fifth outlet 5 with an EP tube to count the flow rate; the concentration, cell survival, cell recovery, and DMSO addition concentration of the cells at the third inlet 3 and the fourth outlet 4 are statistically analyzed by cell counting, fluorescence staining, microscopic observation, and ultraviolet spectrophotometry.

[0062] The test results are as follows: the fourth outlet 4 and the fifth outlet 5 are 0.1 mL / min and 0.4 mL / min respectively; the cell concentration at the fourth outlet 4 is 1×10 6 cells / mL, the cell survival rate is 95%, the cell recovery rate is 90%, and the DMSO addition concentration is 10%.

[0063] In the description of this specification, if there are descriptions of reference terms such as "one embodiment", "some examples", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples", it means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0064] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art.

Claims

1. An upper-layer chip for adding cryoprotectant in multiple stages, characterized in that: The upper-layer chip is provided with a cryoprotectant additive solution generation area (6), and the cryoprotectant additive solution generation area (6) includes a first liquid inlet channel and a first processing unit. The upper-layer chip is provided with a first inlet (1), and the first inlet (1) is set as a through hole penetrating the upper-layer chip. The first inlet (1) is used to introduce a diluent into the first liquid inlet channel. The first processing unit includes a plurality of first mixers arranged in series. The first liquid inlet channel is used to introduce the diluent into each of the first mixers, and each of the first mixers is provided with a first liquid outlet channel; a cryoprotectant stepwise addition area (8), and the cryoprotectant stepwise addition area (8) includes a plurality of second processing units arranged in series. The number of the second processing units is the same as the number of the first mixers. Each of the second processing units is provided with a second liquid inlet and a second liquid outlet. The second liquid inlet is used to connect to the first liquid outlet channel. Each of the second processing units includes a second mixer and a cell separation and concentration module arranged in series. The second liquid inlet is arranged at the inlet of the second mixer, and the second liquid outlet is arranged at the outlet of the cell separation and concentration module; wherein, each of the first mixers in the cryoprotectant additive solution generation area (6) is arranged in reverse order and paired with each of the second processing units in the cryoprotectant stepwise addition area (8). The first liquid outlet channel of the first mixer ranked first in the cryoprotectant additive solution generation area (6) is used to connect to the second liquid inlet of the second processing unit ranked last in the cryoprotectant stepwise addition area (8), and the first liquid outlet channel of the first mixer ranked last in the cryoprotectant additive solution generation area (6) is used to connect to the second liquid inlet of the second processing unit ranked first in the cryoprotectant stepwise addition area (8); The upper-layer chip is provided with a second inlet (2), and the second inlet (2) is used to introduce the cryoprotectant stock solution into the first mixer ranked first in the first processing unit; The upper-layer chip is provided with a third inlet (3), and the third inlet (3) is used to introduce a cell suspension into the cryoprotectant stepwise addition area (8). The third inlet (3) is used to connect to the second liquid inlet of the second processing unit ranked first in the cryoprotectant stepwise addition area (8); The diluent is gradually converged with each of the first mixers in the first processing unit. According to the series order of the first mixers, the concentration of the cryoprotectant additive solution discharged by the later first mixer is lower; In the cryoprotectant stepwise addition area (8), according to the series order of the second processing units, the concentration of the cryoprotectant additive solution introduced into the later second processing unit is higher. The cell suspension introduced through the third inlet (3) flows through the cryoprotectant stepwise addition area (8) step by step, first mixes and reacts with the cryoprotectant additive solution with a low concentration, and the separated and concentrated cell suspension enters the next second processing unit for continuous mixing, separation and concentration.

2. The upper chip with multi-stage addition of cryoprotectant for cells according to claim 1, characterized in that: the upper chip is provided with a waste liquid collection area (9), and each of the second processing units is provided with a second waste liquid discharge channel for communicating with the waste liquid collection area (9), and the second waste liquid discharge channel is arranged at the outlet of the cell separation and concentration module.

3. The upper chip with multi-stage addition of cryoprotectant for cells according to claim 2, characterized in that: the waste liquid collection area (9) is provided with a plurality of bending paths for extending the channel length.

4. The upper chip with multi-stage addition of cryoprotectant for cells according to claim 1, characterized in that: the first liquid inlet channel is provided with a plurality of bending paths for extending the channel length.

5. The upper chip with multi-stage addition of cryoprotectant for cells according to claim 4, characterized in that: the first liquid inlet channel is provided with a plurality of first channels, the number of the first channels is the same as that of the first mixers, each of the first channels is paired with each of the first mixers, and the bending path is arranged in the channel section between two adjacent first channels of the first liquid inlet channel.

6. The upper chip with multi-stage addition of cryoprotectant for cells according to claim 1, characterized in that: each of the first liquid outlet channels is provided with a bending path for extending the channel length.

7. The upper chip with multi-stage addition of cryoprotectant for cells according to claim 1, characterized in that: the upper chip is provided with a fourth outlet (4) for discharging the cell suspension, and the fourth outlet (4) is used for communicating with the second liquid outlet of the second processing unit arranged at the end in the cryoprotectant stepwise addition area (8).

8. A micro-device with multi-stage addition of cryoprotectant for cells, characterized in that: it includes a lower chip and the upper chip according to any one of claims 1 to 7.

Images