A kind of freeze-dried powder of quality control product and its preparation method and application

Abstract

The present invention discloses a quality control product freeze-dried powder and a preparation method and application thereof, and belongs to the technical field of quality control products. The quality control product freeze-dried powder is obtained by freeze-drying a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifugation after slicing, dewaxing and hydration of a paraffin-embedded cell sample. The preparation method of the present invention is used to prepare the quality control product freeze-dried powder, which can reduce the loss of cell fragments during the freeze-drying process, ensure a high recovery rate of cells, and at the same time avoid performance changes or protein loss of cell fragments during the freeze-drying process, and ensure that the immunohistochemical staining performance of the cell precipitate after freeze-drying does not change. The quality control product freeze-dried powder obtained by the preparation method of the present invention has a prolonged storage time and is easy to transport. The cell suspension after redissolution has good dispersibility, retains key target substances, and has normal immunohistochemical staining performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality control products, and in particular, relates to a quality control product freeze-dried powder and a preparation method and application thereof. Background Art

[0002] Liquid cell quality control products contain the target protein and have complete subcellular structures such as cell nucleus, cell membrane, cytoplasm, etc., and can maintain the same expression pattern as the tissue section to be tested in immunohistochemical staining. As a positive control, it can not only show positive staining, but also further control the positive subcellular localization; liquid cell quality control products that do not express the target protein can be used as negative controls. Therefore. Liquid cell quality control products can provide a better basis for judging the results of subsequent immunohistochemical staining.

[0003] However, liquid cell quality control products need to be frozen at -20℃ or transported in refrigerated containers with ice packs, which makes transportation inconvenient. Lyophilized powder cell quality control products provide a better choice. Lyophilized powder cell quality control products can be stored at 2-8℃ and have stable performance. However, current lyophilization technology often leads to low cell recovery rates, which affects test results. Summary of the invention

[0004] In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:

[0005] The first aspect of the present invention provides a quality control freeze-dried powder, which is obtained by freeze-drying a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifuging a paraffin-embedded cell sample after sectioning, dewaxing and hydration.

[0006] In the present invention, the quality control product is a cell quality control product, more specifically, a cell fragment quality control product. In immunoassay, the cell quality control product is a standardized material for calibration and verification of experimental results. They usually contain antigens, antibodies or other immunoreactive components of known quantity and characteristics to ensure the accuracy and repeatability of immunoassay. This quality control product is widely used in immunological tests such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, immunoblotting (Western blot), flow cytometry (Flow Cytometry). In the art, the cell quality control product is generally a liquid quality control product, and the present invention innovatively proposes its lyophilized powder form to extend the protection time without affecting performance.

[0007] In some specific embodiments of the present invention, the method for preparing the cell precipitate is as follows:

[0008] The wax-embedded cell sample is sliced, and the thin slice is placed in a centrifuge tube; a dewaxing solution is poured into the centrifuge tube, and the mixture is shaken to dissolve the paraffin in the slice; the mixture is centrifuged, the dewaxing solution is poured out, and isopropanol is added and the cell precipitation and ethanol are shaken to mix; the mixture is centrifuged, the isopropanol is poured out, and anhydrous ethanol is added to the centrifuge tube, and the mixture is blown evenly with a pipette tip; the mixture is centrifuged, and the cell precipitation is obtained after the ethanol is removed.

[0009] In some embodiments of the present invention, the cell precipitate is freeze-dried in a freeze-drying protective solution, wherein the freeze-drying protective solution includes an excipient, and the excipient includes a stabilizer, a filler, and a buffer.

[0010] Optionally, the stabilizer is selected from at least one of the group consisting of sucrose, lactose, glucose, trehalose, fructose, mannitol, sorbitol, lysine, glycine and serum albumin; optionally, the filler is selected from at least one of the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and derivatives thereof; optionally, the buffer is selected from at least one of the group consisting of phosphate, histidine, glycine, lysine, citrate.

[0011] It is worth noting that some substances can simultaneously act as two or even three of the following: stabilizer, filler, and buffer. For example, sucrose is both a stabilizer and a filler; histidine is both a filler and a buffer; and glycine can be used as a stabilizer, a filler, and a buffer. In this case, those skilled in the art can select the corresponding substances, for example, select sucrose as a stabilizer, and select histidine as both a filler and a buffer.

[0012] In some preferred embodiments of the invention, the stabilizer includes sucrose and glucose. In some more preferred embodiments of the invention, the ratio of sucrose to glucose is 1:4 to 4:1.

[0013] In some preferred embodiments of the present invention, the filler and the buffer are both glycine, histidine or lysine.

[0014] Further, the freeze-drying protective solution also includes a surfactant, and optionally, the surfactant is selected from at least one of the group consisting of Tween-80, Tween-20, polyethylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, Span-80, Span-20 and poloxamer. In some preferred embodiments of the present invention, the surfactant is Tween-80.

[0015] In some specific embodiments of the present invention, the lyophilization protection solution comprises: 3% to 8% stabilizer, 0.5% to 1.5% Tween-80 and 0.3% to 0.5% glycine, wherein the stabilizer is composed of sucrose and glucose, and the ratio of sucrose to glucose is 1:4 to 4:1.

[0016] In some embodiments of the present invention, the freeze-drying is a gradient freeze-drying at -80°C to 0°C.

[0017] In some specific embodiments of the present invention, the -80°C to 0°C gradient freeze-drying is specifically:

[0018] (1) Vacuum degree 100Pa~150Pa, from about -80℃ to about -20℃, keep warm for 1.5~2.5h;

[0019] (2) Vacuum degree 0.02-0.05 mBar, from about -20°C to about -50°C, keep warm for 2.5-3.5 hours;

[0020] (3) from about -50°C to about -30°C, and keep warm for 4.5 to 5.5 hours;

[0021] (4) from about -30°C to about -20°C, keeping warm for 4.5 to 5.5 hours;

[0022] (5) Heat from about -20°C to about 0°C and keep warm for 2.5 to 3.5 hours;

[0023] (6) Heat the temperature from about 0°C to about 25°C and maintain the temperature for 5.5 to 6.5 hours.

[0024] In the present invention, "about" has a clear and specific meaning, and here it refers to the corresponding temperature ±2°C, for example, "about 80°C" means "80±2°C".

[0025] The second aspect of the present invention provides a use of a freeze-dried protective solution in preparing a freeze-dried powder of a cell precipitate, wherein the cell precipitate contains cell fragments as described above, and is obtained by centrifuging a paraffin-embedded cell sample after sectioning, dewaxing and hydration, wherein the freeze-dried protective solution comprises an excipient, and the excipient comprises a stabilizer, a filler and a buffer.

[0026] Optionally, the stabilizer is selected from at least one of the group consisting of sucrose, lactose, glucose, trehalose, fructose, mannitol, sorbitol, lysine, glycine and serum albumin; optionally, the filler is selected from at least one of the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and derivatives thereof; optionally, the buffer is selected from at least one of the group consisting of phosphate, histidine, glycine, lysine, citrate.

[0027] In some preferred embodiments of the invention, the stabilizer includes sucrose and glucose. In some more preferred embodiments of the invention, the ratio of sucrose to glucose is 1:4 to 4:1.

[0028] In some preferred embodiments of the present invention, the filler and the buffer are both glycine, histidine or lysine.

[0029] Further, the freeze-drying protective solution also includes a surfactant, and optionally, the surfactant is selected from at least one of the group consisting of Tween-80, Tween-20, polyethylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, Span-80, Span-20 and poloxamer. In some preferred embodiments of the present invention, the surfactant is Tween-80.

[0030] In some specific embodiments of the present invention, the lyophilization protection solution comprises: 3% to 8% stabilizer, 0.5% to 1.5% Tween-80 and 0.3% to 0.5% glycine, wherein the stabilizer is composed of sucrose and glucose, and the ratio of sucrose to glucose is 1:4 to 4:1.

[0031] The third aspect of the present invention provides a cell suspension, which is obtained by mixing the freeze-dried protective solution described in any one of the second aspects of the present invention and a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifuging a paraffin-embedded cell sample after sectioning, dewaxing and hydration.

[0032] The cell suspension is a cell suspension before freeze-drying. In some specific embodiments of the present invention, each 1 mL of the cell suspension contains 2.5M to 4.2M cell fragments.

[0033] The fourth aspect of the present invention provides another cell suspension, which is obtained by reconstituting any of the freeze-dried powders of the quality control product described in the first aspect of the present invention with a solvent.

[0034] The cell suspension can be directly used for immunoassay. In some embodiments of the present invention, the solvent can be any solvent that can disperse the cell fragments. In some specific embodiments of the present invention, the solvent is selected from one of water, alcohol of any concentration, and a semi-solid buffer. The semi-solid buffer includes a water retainer and a preservative. The water retainer is polyethylene glycol with a volume fraction of 10%, and the preservative is arginine with a volume fraction of 1% and sodium azide with a volume fraction of 0.01%.

[0035] The fifth aspect of the present invention provides a method for preparing the freeze-dried powder of the quality control product described in the first aspect of the present invention, comprising the step of performing gradient freeze-drying of the cell precipitate at -80°C to 0°C.

[0036] In some specific embodiments of the present invention, the -80°C to 0°C gradient freeze-drying is specifically:

[0037] (1) Vacuum degree 100Pa~150Pa, from about -80℃ to about -20℃, keep warm for 1.5~2.5h;

[0038] (2) Vacuum degree 0.02-0.05 mBar, from about -20°C to about -50°C, keep warm for 2.5-3.5 hours;

[0039] (3) from about -50°C to about -30°C, and keep warm for 4.5 to 5.5 hours;

[0040] (4) from about -30°C to about -20°C, keeping warm for 4.5 to 5.5 hours;

[0041] (5) Heat from about -20°C to about 0°C and keep warm for 2.5 to 3.5 hours;

[0042] (6) Heat the temperature from about 0°C to about 25°C and maintain the temperature for 5.5 to 6.5 hours.

[0043] In the present invention, "about" has a clear and specific meaning, and here it refers to the corresponding temperature ±2°C, for example, "about 80°C" means "80±2°C".

[0044] The sixth aspect of the present invention provides the use of any of the lyophilized powders of the quality control product described in the first aspect of the present invention in the preparation of a kit for immunoassay, wherein the immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, Western blot, and flow cytometry.

[0045] Beneficial effects of the present invention

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The present invention can disperse cell fragments during the quick freezing process and prevent cell fragments from agglomerating by optimizing the components and proportions of the carbohydrate protective agent. Furthermore, by selecting the surfactant and the dosage, the polarity of the cells changes slowly during the solvent replacement process, so that the cells are not easily adhered to the container, the loss of cell fragments during the freeze-drying process is reduced, and the freeze-drying recovery rate is improved. Finally, by selecting the buffer and the dosage, the effective components in the cell fragments can be protected.

[0048] The method for preparing the freeze-dried powder of the quality control product of the present invention only takes about 24 hours, the freeze-drying time is greatly shortened, and the loss of cell fragments during the freeze-drying process can be reduced, ensuring a high recovery rate of the cell fragments, while avoiding performance changes or protein loss in the cells during the freeze-drying process, and ensuring that the cell precipitate is re-dissolved after freeze-drying to retain key target substances, and the immunohistochemical staining performance is not changed.

[0049] The quality control product freeze-dried powder of the present invention has a prolonged storage time and is convenient for transportation; the cell suspension after reconstitution has good dispersibility and normal immunohistochemical staining performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The cell dispersion of the lyophilized powder of the liquid SK-OV-3 cell quality control prepared by using different lyophilization protection solutions after reconstitution is shown, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyophilization protection solution #1; D: reconstitution after lyophilization with lyophilization protection solution #2; E: reconstitution after lyophilization with lyophilization protection solution #3; F: reconstitution after lyophilization with lyophilization protection solution #4; G: reconstitution after lyophilization with lyophilization protection solution #5.

[0051] Figure 2 The immunohistochemical staining performance of the corresponding target CK7 after reconstitution of the liquid SK-OV-3 cell quality control freeze-dried powder prepared using different freeze-dried protective solutions is shown, A: cell suspension before freeze-drying; B: reconstitution after freeze-drying with ddH2O; C: reconstitution after freeze-drying with freeze-dried protective solution #1; D: reconstitution after freeze-drying with freeze-dried protective solution #2; E: reconstitution after freeze-drying with freeze-dried protective solution #3; F: reconstitution after freeze-drying with freeze-dried protective solution #4; G: reconstitution after freeze-drying with freeze-dried protective solution #5.

[0052] Figure 3 The cell dispersion of the liquid Molt-4 cell quality control freeze-dried powder prepared with freeze-drying protection solution #1 after reconstitution with different solvents is shown, A: cell suspension before freeze-drying; B: reconstitution with water; C: reconstitution with 25% alcohol; D: reconstitution with semi-solid buffer.

[0053] Figure 4 The immunohistochemical staining performance of the corresponding target CD3 of the liquid Molt-4 cell quality control freeze-dried powder prepared by freeze-drying protection solution #1 after reconstitution with different solvents is shown, A: cell suspension before freeze-drying; B: reconstitution with water; C: reconstitution with 25% alcohol; D: reconstitution with semi-solid buffer.

[0054] Figure 5 The cell dispersion after reconstitution of the liquid Molt-4 cell quality control freeze-dried powder prepared with freeze-dried protective solution #1 and ddH2O, respectively, is shown. A: cell suspension before freeze-drying; B: reconstitution after freeze-drying with ddH2O; C: reconstitution after freeze-drying with freeze-dried protective solution #1.

[0055] Figure 6 The immunohistochemical staining performance of the corresponding target CD3 after reconstitution of the liquid Molt-4 cell quality control freeze-dried powder prepared by freeze-dried protective solution #1 and ddH2O, respectively, is shown. A: cell suspension before freeze-drying; B: reconstitution after freeze-drying with ddH2O; C: reconstitution after freeze-drying with freeze-dried protective solution #1.

[0056] Figure 7 The cell dispersion after reconstitution of the liquid Ramos cell quality control freeze-dried powder prepared with freeze-dried protective solution #1 and ddH2O, respectively, is shown. A: cell suspension before freeze-drying; B: reconstitution after freeze-drying with ddH2O; C: reconstitution after freeze-drying with freeze-dried protective solution #1.

[0057] Figure 8 The immunohistochemical staining performance of the corresponding target CD20 after reconstitution of the liquid Ramos cell quality control freeze-dried powder prepared with freeze-dried protective solution #1 and ddH2O, respectively, is shown. A: cell suspension before freeze-drying; B: reconstitution after freeze-drying with ddH2O; C: reconstitution after freeze-drying with freeze-dried protective solution #1. DETAILED DESCRIPTION

[0058] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0059] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include numerical values ​​outside the range. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range comprising a numerical value less than 1 or a range comprising a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For a range comprising a single digit less than 10 (e.g., 1 to 5), 1 unit is generally considered as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of numerical values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0060] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0061] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0062] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0064] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0065] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0066] Example 1: Freeze-dried liquid for liquid cell quality control

[0067] ddH2O was used as a solvent, and 2% sucrose, 3% glucose, 1% Tween-80 and 0.5% glycine were added to obtain a freeze-dried protective solution #1 for preparing a liquid cell quality control freeze-dried powder.

[0068] In order to demonstrate the excellent effect of the above freeze-dried protective solution, the inventors also prepared a freeze-dried protective solution with the following formula.

[0069] Lyophilization protection solution #2: ddH2O as solvent, 5% sucrose, 1% Tween-80 and 0.5% glycine;

[0070] Freeze-dried protective solution #3: ddH2O was used as the solvent, and 2% sucrose, 3% trehalose, 1% Tween-80 and 0.5% glycine were added.

[0071] Lyophilization protection solution #4: ddH2O was used as the solvent, and 4% sucrose, 1% glucose, 1% Tween-80 and 0.5% glycine were added.

[0072] Lyophilization protection solution #5: ddH2O was used as the solvent, and 1% sucrose, 4% glucose, 1% Tween-80 and 0.5% glycine were added.

[0073] Example 2 Preparation process of lyophilized powder of liquid cell quality control product

[0074] 1. Preparation of Cell Suspension

[0075] The cell suspension was prepared according to the method of Example 1 in Chinese invention patent CN112611614B - "A method for preparing a semi-solid cell suspension and its application":

[0076] The wax-embedded cell samples were sliced ​​and the thin slices were placed in a centrifuge tube; about 25 mL of dewaxing solution was poured into the centrifuge tube and vortexed to dissolve the paraffin in the slices; the mixture was centrifuged at 2500 rpm for 5 min, the dewaxing solution was discarded, 25 mL of isopropanol was added and the cell pellet and ethanol were vortexed to mix; the mixture was centrifuged at 2500 rpm for 5 min, the isopropanol was discarded, and about 1 mL of anhydrous ethanol was added to the centrifuge tube and pipetted evenly; the mixture was placed in a 1.5 mL centrifuge tube and centrifuged at 2500 rpm for 5 min, and the ethanol was removed with a pipette.

[0077] 1 mL of freeze-drying protection solution was added to the cell precipitate containing 2.5 M to 4.2 M cell debris to obtain a cell suspension to be freeze-dried.

[0078] 2. Packaging

[0079] The cell suspension obtained in step 1 was dispensed into freeze-drying containers, with each portion being 0.3 mL.

[0080] 3. Quick freezing

[0081] The cell suspension after the subpackaging in step 2 and the freeze-drying container are placed in a low temperature environment below -80°C for quick freezing;

[0082] 4. Freeze Drying

[0083] First, the in-situ freeze dryer (Mercury 2.5L, Hangzhou Fu Ruijie Technology Co., Ltd.) was precooled to below -50°C, and the solid obtained by quick freezing in step 3 was heated together with the freeze drying container. The drying was divided into 6 stages:

[0084] (7) The vacuum degree is controlled at 100 Pa 150 Pa, the temperature is raised from -80 °C to -20 °C, and the temperature is kept for 2 h;

[0085] (8) Evacuate; the vacuum degree is reduced to 0.04 mBar within 5 minutes, and the temperature is reduced from -20°C to -50°C, and kept for 3 hours;

[0086] (9) From -50°C to -30°C, keep warm for 5 hours;

[0087] (10) From -30°C to -20°C, keep warm for 5 hours;

[0088] (11) Heat from -20°C to 0°C and keep warm for 3 hours;

[0089] (12) The temperature was raised from 0°C to 25°C and kept at this temperature for 6 h to complete the freeze-drying process and form a freeze-dried powder.

[0090] 5.Save

[0091] The cell quality control freeze-dried powder harvested in step 4 is sealed and packaged.

[0092] Example 3 Performance of cell quality control freeze-dried powder prepared with different freeze-drying protection solutions

[0093] The liquid SK-OV-3 cell quality control product was freeze-dried using freeze-dried protective solutions #1 to #5 and ddH2O (without adding protective components) in Example 1 according to the method of Example 2 to obtain different freeze-dried powders of SK-OV-3 cell quality control products.

[0094] The SK-OV-3 cell quality control freeze-dried powder prepared with different freeze-dried protective solutions was reconstituted with 25% alcohol and spotted using the fully automatic slide processing system Plus60 of Tuling (Hangzhou) Biotechnology Co., Ltd. After drying, immunohistochemistry experiments were performed to verify the staining performance, and the liquid SK-OV-3 cell quality control before freeze-drying was used as a control.

[0095] Immunohistochemistry method: The experiment was carried out using the LYNX480 fully automatic immunohistochemistry staining machine produced by Toulin (Hangzhou) Biotechnology Co., Ltd. The target primary antibody, secondary antibody, auxiliary reagents, etc. used in conjunction were purchased from the commercialized kits of Toulin (Hangzhou) Biotechnology Co., Ltd. The experiment adopted the conventional immunohistochemistry experimental process and was performed according to the instructions of the kit.

[0096] After the freeze-dried powder of SK-OV-3 cell quality control prepared by freeze-dried protective solution was reconstituted, it was first stained with hematoxylin and returned to blue to observe the cell dispersion. The results are as follows Figure 1 As shown. Figure 1 It can be seen that for the freeze-dried samples using ddH2O (without adding protective ingredients) as the solvent, the cell fragments are stacked and unevenly dispersed after reconstitution, and the density is significantly reduced and there is loss; for the freeze-dried samples using a single sugar (freeze-dried protective solution #2) and glucose replaced with trehalose (freeze-dried protective solution #3), the cell fragments are slightly stacked and slightly unevenly dispersed after reconstitution; for the freeze-dried samples using the other formulas (freeze-dried protective solutions #1, #4, and #5), the cell fragments are rarely stacked and evenly dispersed after reconstitution.

[0097] The results of immunohistochemical staining performance verification of CK7 corresponding to the target of SK-OV-3 cells are as follows Figure 2 As shown, it can be seen that the immunohistochemical staining is unclear due to cell stacking when ddH2O (no protective component added) is used as the solvent for freeze-drying; the staining performance of the freeze-dried solution with added protective agent but using a single sugar (freeze-dried protective solution #2) and glucose replaced with trehalose (freeze-dried protective solution #3) is slightly worse; the staining performance of the remaining formulas (freeze-dried protective solutions #1, #4, #5) is basically the same as before freeze-drying.

[0098] The above results all show that the cells were evenly dispersed and the staining performance was good when lyophilized powder of SK-OV-3 cell quality control was prepared using lyophilized protective solution #1, #4, and #5. In addition, the staining effect of SK-OV-3 cell quality control lyophilized powder prepared with lyophilized protective solution #1 was the best after reconstitution.

[0099] The inventors further replaced the surfactant Tween-80 with Tween-20, or replaced the buffer and filler glycine with histidine or lysine, which can also function as a buffer and filler, on the basis of lyophilized protective solution #1. They found that after the prepared lyophilized powder of the quality control product was reconstituted, the cells were still very evenly dispersed and the staining performance did not decrease significantly.

[0100] The inventors also found that the sucrose concentration is 1-4% (i.e., the ratio of sucrose to glucose is 1:4-4:1), the surfactant (Tween-80, Tween-20, polyethylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, Span-80, Span-20 or poloxamer) concentration is 0.5-1%, and the buffer (glycine, histidine or lysine) concentration is 0.3-0.5%, all of which do not affect the cell dispersion effect and staining performance.

[0101] Example 4: Lyophilized powder of cell quality control prepared by conventional lyophilization process

[0102] In order to verify the effect of the gradient freeze-drying process of Example 2 of the present invention, the inventors used the freeze-drying protection solutions #1 to #5 in Example 1 and ddH2O (without adding protection components) to freeze-dry the liquid SK-OV-3 cell quality control product according to the conventional freeze-drying method.

[0103] The conventional freeze-drying method is as follows:

[0104] The cell suspension was placed in a -80°C environment for low-temperature quick freezing, and placed together with the freeze-drying container in a pre-cooled freeze dryer. The partitions were not heated or insulated, and vacuum operation was directly performed at room temperature. The vacuum degree dropped to 0.04 mBar within 5 minutes, and the vacuum state was maintained for 36 hours to complete the freeze-drying operation.

[0105] The results showed that no matter which freeze-drying protection solution was used, the cells of the obtained freeze-dried powder were stacked together after reconstitution and could not be dispersed and could not be used for detection. This shows that the freeze-drying protection solution of the present invention needs to be used in conjunction with the freeze-drying process of Example 2 of the present invention to obtain the freeze-dried powder of the quality control product with excellent performance.

[0106] Example 5 Effect of different reconstitution solvents on test results

[0107] The liquid Molt-4 cell quality control product was freeze-dried using the freeze-drying protection solution #1 in Example 1 according to the method of Example 2 to obtain a Molt-4 cell quality control product freeze-dried powder.

[0108] The Molt-4 cell control freeze-dried powder was reconstituted using the following three solvents:

[0109] (1) Water;

[0110] (2) 25% alcohol;

[0111] (3) The semi-solid buffer solution in CN112611614B includes a water retaining agent and a preservative, wherein the water retaining agent is polyethylene glycol with a volume fraction of 10%, the preservative is arginine with a volume fraction of 1% and sodium azide with a volume fraction of 0.01%, and also includes eosin solution with a volume fraction of 0.1%.

[0112] After the Molt-4 cell quality control freeze-dried powder was reconstituted with different solvents, it was first stained with hematoxylin and returned to blue to observe the cell dispersion. The results were as follows Figure 3 As shown. Figure 3 It can be seen that the cell fragments are evenly dispersed when reconstituted with different solvents and are basically the same as before freeze-drying.

[0113] The results of immunohistochemical staining performance verification of Molt-4 cell corresponding target CD3 are as follows Figure 4 As shown, it can be seen that the immunohistochemical staining performance is good when reconstituted with different solvents and is basically consistent with that before freeze-drying.

[0114] In summary, after using the freeze-drying process of Example 2 of the present invention, no matter which solvent is used for reconstitution, the cell dispersion and staining performance are not much different, and both meet the requirements of the detection.

[0115] Example 6 Freeze-drying effect of freeze-drying protection solution #1 on liquid Molt-4 cell quality control

[0116] The liquid Molt-4 cell quality control product was freeze-dried using the freeze-drying protection solution #1 and ddH2O (without adding protection components) in Example 1 according to the method of Example 2 to obtain a Molt-4 cell quality control product freeze-dried powder.

[0117] After the freeze-dried powder of Molt-4 cell quality control was reconstituted with 25% alcohol, it was first stained with hematoxylin and returned to blue to observe the cell dispersion and compared with the cell suspension before freeze-drying. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that when freeze-dried using ddH2O (without adding protective ingredients) as the solvent, the cell fragments are stacked, dispersed unevenly, and the density is significantly reduced, with loss; when freeze-dried using freeze-drying protective solution #1, the cell fragments are rarely stacked and dispersed evenly.

[0118] The results of immunohistochemical staining performance verification of Molt-4 cell corresponding target CD3 are as follows Figure 6 As shown, it can be seen that when ddH2O (without added protective ingredients) is used as the solvent for freeze-drying, the immunohistochemical staining is unclear due to cell stacking; when freeze-dried using freeze-drying protective solution #1, the staining performance is basically the same as before freeze-drying.

[0119] In summary, the liquid Molt-4 cell quality control product was freeze-dried using the freeze-drying protection solution #1 in Example 1, and the uniformity of cell fragments and the staining performance of the obtained Molt-4 cell quality control freeze-dried powder after reconstitution were basically the same as before freeze-drying; in contrast, the liquid Molt-4 cell quality control product was freeze-dried using ddH2O, and the cell fragments of the obtained Molt-4 cell quality control freeze-dried powder were uneven and the staining performance was poor after reconstitution.

[0120] Example 7 Freeze-drying effect of freeze-drying protection solution #1 on liquid Ramos cell quality control product

[0121] The liquid Ramos cell quality control product was freeze-dried using the freeze-drying protection solution #1 and ddH2O (without adding protection components) in Example 1 according to the method of Example 2 to obtain a Ramos cell quality control product freeze-dried powder.

[0122] After the Ramos cell quality control freeze-dried powder was reconstituted with 25% alcohol, it was first stained with hematoxylin and returned to blue to observe the cell dispersion and compared with the cell suspension before freeze-drying. The results are as follows: Figure 7 As shown. Figure 7 It can be seen that when freeze-dried using ddH2O (without adding protective ingredients) as the solvent, the cell fragments are stacked, dispersed unevenly, and the density is significantly reduced, with loss; when freeze-dried using freeze-drying protective solution #1, the cell fragments are rarely stacked and dispersed evenly.

[0123] The results of immunohistochemical staining performance verification of the target CD20 of Ramos cells are as follows Figure 8 As shown, it can be seen that when ddH2O (without added protective ingredients) is used as the solvent for freeze-drying, the immunohistochemical staining is unclear due to cell stacking; when freeze-dried using freeze-drying protective solution #1, the staining performance is basically the same as before freeze-drying.

[0124] In summary, the liquid Ramos cell quality control product was freeze-dried using the freeze-drying protection solution #1 in Example 1, and the uniformity of cell fragments and the staining performance of the obtained Ramos cell quality control freeze-dried powder after reconstitution were basically the same as before freeze-drying; comparatively speaking, the liquid Ramos cell quality control product was freeze-dried using ddH2O, and the cell fragments of the obtained Ramos cell quality control freeze-dried powder were uneven and the staining performance was poor after reconstitution.

[0125] Example 8: Recovery rate experiment of freeze-dried liquid Molt-4 cell quality control product

[0126] The liquid Molt-4 cell quality control product was used to verify the freeze-drying recovery rate. 4.0 mL of qualified liquid Molt-4 cell quality control product was taken, and the solvent was replaced and divided into 8 portions. Four portions each were freeze-dried using freeze-drying protection liquid #1 and ddH2O (no protective component was added). The products were pre-frozen at -80°C and freeze-dried. The process adopted the conditions and parameters of Example 2. After freeze-drying, 8 bottles of Molt-4 cell quality control freeze-dried powder were obtained.

[0127] After fully re-dissolving with 500 μL of 25% alcohol, count by flow cytometry (MAC&Quant Analyzer), take 50 μL of the re-dissolved solution and centrifuge. Discard the supernatant, add 1 mL of 1×PBS to resuspend, dilute 20 times and take 20 μL for FACS loading until the sample is measured. The number of Molt-4 cell fragments A in the 20 μL sample is obtained, and the above operation is repeated to detect the number of Molt-4 cell fragments B before freeze-drying, and the freeze-drying recovery rate is calculated by substituting into the following formula.

[0128] Recovery rate % = A / B

[0129] The results are shown in Table 1.

[0130] Table 1 Recovery results of lyophilized liquid Molt-4 cell quality control products

[0131]

[0132] Note: The control is the cell count result of the liquid Molt-4 cell quality control before lyophilization.

[0133] As shown in Table 1, the recovery rate of the liquid Molt-4 cell quality control product freeze-dried with freeze-drying protection solution #1 exceeded 70%, and reached more than 80% at most; while the recovery rate of the liquid Molt-4 cell quality control product freeze-dried with ddH2O was only 35%-40%.

[0134] Example 9: Recovery rate experiment of freeze-dried liquid Ramos cell quality control product

[0135] The liquid Ramos cell quality control product was used to verify the freeze-drying recovery rate. 4.0 mL of qualified liquid Ramos cell quality control product was taken, and the solvent was replaced and divided into 8 portions. Four portions each were freeze-dried using freeze-drying protection solution #1 and ddH2O (without adding protection components). The products were pre-frozen at -80°C and freeze-dried. The process adopted the conditions and parameters of Example 2. After freeze-drying, 8 bottles of Ramos cell quality control freeze-dried powder were obtained.

[0136] After being fully reconstituted with 500 μL of 25% alcohol, the same counting method as in Example 7 was used to count and calculate the freeze-dried recovery rate. The results are shown in Table 2.

[0137] Table 2 Recovery results of liquid Ramos cell quality control freeze-dried products

[0138]

[0139] Note: The control is the cell count result of the liquid Ramos cell quality control before lyophilization.

[0140] As shown in Table 1, the recovery rate of the liquid Ramos cell quality control product freeze-dried with freeze-drying protection solution #1 exceeded 70%, and the highest exceeded 80%; while the recovery rate of the liquid Ramos cell quality control product freeze-dried with ddH2O was only 30%-40%.

[0141] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A quality control freeze-dried powder, characterized in that: The control freeze-dried powder is obtained by freeze-drying a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifuging a paraffin-embedded cell sample for immunohistochemical detection after sectioning, dewaxing and hydration. The cell precipitate is freeze-dried in a freeze-drying protective solution, wherein the freeze-drying protective solution includes an excipient and a surfactant, and the excipient includes a stabilizer, a filler and a buffer. The stabilizer includes sucrose and glucose, and the ratio of sucrose to glucose is 1:4 to 4:

1. The freeze-drying is -80°C ~ 0°C gradient freeze-drying, and the -80°C ~ 0°C gradient freeze-drying is specifically: (1) Vacuum degree 100Pa~150Pa, from about -80℃ to about -20℃, keep warm for 1.5~2.5h; (2) Vacuum degree 0.02~0.05mBar, drop from about -20℃ to about -50℃, keep warm for 2.5~3.5h; (3) From about -50°C to about -30°C, keep warm for 4.5-5.5 hours; (4) From about -30°C to about -20°C, keep warm for 4.5-5.5 hours; (5) From about -20°C to about 0°C, keep warm for 2.5 to 3.5 hours; (6) Raise the temperature from about 0°C to about 25°C and keep warm for 5.5 to 6.5 hours.

2. The quality control freeze-dried powder according to claim 1, characterized in that: The filler is selected from at least one of the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and derivatives thereof; the buffer is selected from at least one of the group consisting of phosphate, histidine, glycine, lysine and citrate.

3. The quality control freeze-dried powder according to claim 1 or 2, characterized in that: The surfactant is at least one selected from the group consisting of Tween-80, Tween-20, polyethylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, Span-80, Span-20 and poloxamer.

4. Use of a freeze-dried protective solution in the preparation of a quality control freeze-dried powder as claimed in claim 1, characterized in that: The cell precipitate contains cell fragments and is obtained by centrifuging a paraffin-embedded cell sample for immunohistochemical detection after sectioning, dewaxing and hydration. The freeze-drying protective solution includes an excipient and a surfactant. The excipient includes a stabilizer, a filler and a buffer. The stabilizer includes sucrose and glucose, and the ratio of sucrose to glucose is 1:4~4:1; the filler is selected from at least one of the group including sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and its derivatives; the buffer is selected from at least one of the group including phosphate, histidine, glycine, lysine and citrate, and the freeze-drying is a gradient freeze-drying at -80°C~0°C.

5. The use according to claim 4, characterized in that: The surfactant is at least one selected from the group consisting of Tween-80, Tween-20, polyethylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, Span-80, Span-20 and poloxamer.

6. The use according to claim 5, characterized in that: The freeze-dried protective solution includes 3% to 8% stabilizer, 0.5 to 1.5% Tween-80 and 0.3 to 0.5% glycine.

7. A cell suspension, characterized in that The control product lyophilized powder is obtained by reconstituted with a solvent.

8. Use of the freeze-dried powder of the quality control product according to any one of claims 1 to 3 in the preparation of a kit for immunoassay, characterized in that: The immunodetection is selected from the group consisting of immunohistochemistry, immunocytochemistry, and in situ hybridization.

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