Quality control material freeze-dried powder as well as preparation method therefor and use thereof

Abstract

The present application belongs to the technical field of quality control materials. Disclosed are a quality control material freeze-dried powder as well as a preparation method therefor and the use thereof. The quality control material freeze-dried powder is obtained by freeze-drying cell pellets containing cell fragments, the cell pellets being obtained by sectioning, deparaffinizing and hydrating a paraffin-embedded cell sample and then centrifuging same. The quality control material freeze-dried powder prepared by using the preparation method in the present application can reduce the loss of cell fragments during the freeze-drying process, so as to ensure the high recovery rate of cells, can avoid performance change or protein loss of cell fragments during the freeze-drying process, and can ensure that the immunohistochemical staining performance of reconstituted cell pellets after freeze-drying remains unchanged. The quality control material freeze-dried powder obtained by using the preparation method in the present application has extended storage time and facilitates transport, and the reconstituted cell suspension has good dispersibility, retains key target substances, and exhibits normal immunohistochemical staining performance.

Description

A quality control product freeze-dried powder, its preparation method and application TECHNICAL FIELD

[0001] The present application belongs to the technical field of quality control products, and specifically relates to a quality control product freeze-dried powder, its preparation method and application. BACKGROUND

[0002] The liquid cell quality control product contains a target protein and has complete subcellular structures such as a cell nucleus, a cell membrane and a cytoplasm, and can keep a consistent expression pattern with a tissue section to be detected in immunohistochemical staining. As a positive control, the liquid cell quality control product can not only show positive staining but also make further control on positive subcellular localization; the liquid cell quality control product without expression of the target protein can be used as a negative control. Therefore, the liquid cell quality control product can provide a better judgment basis for subsequent immunohistochemical staining results.

[0003] However, the liquid cell quality control product needs to be stored at -20 DEG C or transported with ice bags, causing inconvenient transportation. The cell quality control product in the form of freeze-dried powder provides a better choice, and the cell quality control product freeze-dried powder can be stored at 2-8 DEG C and has stable performance, but the current freeze-drying technology often leads to low cell recovery rate, affecting the detection result. SUMMARY

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

[0005] The first aspect of the present application provides a quality control product freeze-dried powder, which is obtained by freeze-drying of a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifugation after slicing, deparaffinization and hydration treatment of a paraffin-embedded cell sample.

[0006] In the present application, the quality control product is a cell quality control product, more specifically, a cell fragment quality control product. In immunodetection, the cell quality control product is a standardized material used for calibrating and verifying experimental results. They usually contain known quantities and characteristics of antigens, antibodies or other immunoreactive components, ensuring the accuracy and repeatability of immunodetection. Such quality control products are widely used in immunological detection such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, immunoblotting (Western blot), flow cytometry (Flow Cytometry) and the like. In the field, the cell quality control product is generally a liquid quality control product, and the present application innovatively proposes its freeze-dried powder form, which prolongs the protection time without affecting the performance.

[0007] In some specific embodiments of the present application, the preparation method of the cell precipitate is as follows:

[0008] The cell sample embedded in wax block is sliced, and the thin layer is placed in a centrifuge tube; about 5ml of xylene is poured into the centrifuge tube, and the mixture is shaken to dissolve the paraffin in the slice; the mixture is centrifuged, and the xylene is poured out; after isopropanol is added, the cell precipitate is shaken with ethanol; the mixture is centrifuged, and the isopropanol is poured out; anhydrous ethanol is added to the centrifuge tube, and the mixture is blown with a pipette; the mixture is centrifuged, and the ethanol is removed to obtain the cell precipitate.

[0009] In some embodiments of the present application, the cell precipitate is freeze-dried in a freeze-drying protective solution, wherein the freeze-drying protective solution comprises excipients, and the excipients comprise stabilizers, fillers, and buffers.

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

[0011] It is worth noting that for some substances, two or even three can simultaneously act as stabilizers, fillers, and buffers, for example, sucrose is both a stabilizer and a filler; for example, histidine is both a filler and a buffer; for example, glycine can be both a stabilizer and a filler, and can also be a buffer. In this case, the skilled person can select the corresponding substances, for example, sucrose as a stabilizer, and histidine as a filler and a buffer.

[0012] In some preferred embodiments of the present application, the stabilizers comprise sucrose and glucose. In some more preferred embodiments of the present application, the ratio of sucrose and glucose is 1:4 to 4:1.

[0013] In some preferred embodiments of the present application, the fillers and buffers are both glycine, histidine, or lysine.

[0014] Further, the freeze-drying protective solution further comprises a surfactant, and optionally, 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. In some preferred embodiments of the present application, the surfactant is Tween-80.

[0015] In some embodiments of the present application, the freeze-drying protective solution comprises 3% to 8% stabilizer, 0.5% to 1.5% Tween-80 and 0.3% to 0.5% glycine, wherein the stabilizer consists of sucrose and glucose, and the ratio of sucrose to glucose is 1:4 to 4:1.

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

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

[0018] (1) the vacuum degree is 100 Pa to 150 Pa, from about -80°C to about -20°C, and the temperature is kept for 1.5 to 2.5 h;

[0019] (2) the vacuum degree is 0.02 to 0.05 mBar, from about -20°C to about -50°C, and the temperature is kept for 2.5 to 3.5 h;

[0020] (3) from about -50°C to about -30°C, and the temperature is kept for 4.5 to 5.5 h;

[0021] (4) from about -30°C to about -20°C, and the temperature is kept for 4.5 to 5.5 h;

[0022] (5) from about -20°C to about 0°C, and the temperature is kept for 2.5 to 3.5 h;

[0023] (6) from about 0°C to about 25°C, and the temperature is kept for 5.5 to 6.5 h.

[0024] In the present application, "about" has a specific meaning, which here refers to the corresponding temperature ± 2°C, for example, "about 80°C" refers to "80 ± 2°C".

[0025] The second aspect of the present application provides a use of a freeze-drying protective solution in the preparation of a freeze-dried powder of a cell precipitate, wherein the cell precipitate contains cell fragments as described above, and is obtained by centrifugation after the treatment of a paraffin-embedded cell sample by slicing, deparaffinization and hydration, and the freeze-drying protective solution comprises an excipient, wherein the excipient comprises a stabilizer, a bulking agent and a buffer, optionally, the stabilizer is at least one selected from the group consisting of sucrose, lactose, glucose, trehalose, fructose, mannitol, sorbitol, lysine, glycine and serum albumin; optionally, the bulking agent is at least one selected from the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and its derivatives; and optionally, the buffer is at least one selected from the group consisting of phosphate, histidine, glycine, lysine and citrate.

[0026] In some preferred embodiments of the application, the stabilizer comprises sucrose and glucose. In some more preferred embodiments of the application, the ratio of sucrose and glucose is 1:4~4:1.

[0027] In some preferred embodiments of the application, both the bulking agent and the buffering agent are glycine, histidine or lysine.

[0028] Further, the lyoprotectant solution further comprises a surfactant, optionally, 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. In some preferred embodiments of the application, the surfactant is Tween-80.

[0029] In some specific embodiments of the application, the lyoprotectant solution comprises 3%~8% stabilizer, 0.5%~1.5% Tween-80 and 0.3%~0.5% glycine, wherein the stabilizer consists of sucrose and glucose, and the ratio of sucrose and glucose is 1:4~4:1.

[0030] The third aspect of the application provides a cell suspension, which is obtained by mixing the lyoprotectant solution of any one of the second aspect of the application and a cell pellet containing cell fragments, wherein the cell pellet is obtained by centrifugation after slicing, deparaffinization and hydration treatment of a paraffin-embedded cell sample.

[0031] The cell suspension is a cell suspension before lyophilization, and in some specific embodiments of the application, 2.5M~4.2M cell fragments are contained in 1mL of the cell suspension.

[0032] The fourth aspect of the application provides another cell suspension, which is obtained by reconstituting the lyophilized powder of quality control of any one of the first aspect of the application with a solvent.

[0033] The cell suspension is a cell suspension that can be directly used for immunodetection, and in some embodiments of the application, the solvent can be any solvent that can obtain the dispersion of cell fragments. In some specific embodiments of the application, the solvent is selected from one of water, any concentration of alcohol and semi-solid liquid buffer. The semi-solid buffer comprises a water-retaining agent and a preservative, the water-retaining agent is 10% polyethylene glycol by volume fraction, and the preservative is 1% arginine by volume fraction and 0.01% sodium azide by volume fraction.

[0034] The fifth aspect of the application provides a preparation method of the lyophilized powder of quality control of the first aspect of the application, which comprises the step of gradient lyophilization of the cell pellet at-80℃~0℃.

[0035] In some embodiments of the present application, the -80℃~0℃ gradient freeze-drying is specifically:

[0036] (1) The vacuum degree is 100 Pa~150 Pa, from about -80℃ to about -20℃, and the temperature is kept for 1.5~2.5 h;

[0037] (2) The vacuum degree is 0.02~0.05 mBar, from about -20℃ to about -50℃, and the temperature is kept for 2.5~3.5 h;

[0038] (3) From about -50℃ to about -30℃, and the temperature is kept for 4.5~5.5 h;

[0039] (4) From about -30℃ to about -20℃, and the temperature is kept for 4.5~5.5 h;

[0040] (5) From about -20℃ to about 0℃, and the temperature is kept for 2.5~3.5 h;

[0041] (6) From about 0℃ to about 25℃, and the temperature is kept for 5.5~6.5 h.

[0042] In the present application, "about" has a specific meaning, which here refers to the corresponding temperature ±2℃, for example, "about 80℃" refers to "80±2℃".

[0043] The sixth aspect of the present application provides the use of the quality control product freeze-dried powder of any one of the first aspect of the present application in the preparation of a kit for immunodetection, wherein the immunodetection is selected from one of the group consisting of enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, immunoblotting (Western blot), flow cytometry.

[0044] Advantages of the present application

[0045] Compared with the prior art, the present application has the following advantages:

[0046] The present application can disperse cell fragments during the quick-freezing process by optimizing the components and proportions of the sugar protective agent, thereby avoiding the formation of cell fragments into clusters. Further, by screening the surfactant and the amount thereof, the polarity of the cells changes slowly during the solvent replacement process, thereby reducing the adhesion of the cells to the container, reducing the loss of cell fragments during the freeze-drying process, and improving the freeze-drying recovery rate. Finally, by screening the buffer and the amount thereof, the effective components in the cell fragments can be protected.

[0047] The method for preparing the lyophilized powder of the quality control product of the application only needs about 24 hours, the lyophilization time is greatly shortened, the loss of cell fragments during lyophilization can be reduced, the high recovery rate of cell fragments is ensured, the performance change or protein loss of cells during lyophilization can be avoided, and the key target substance can be reserved after the cell precipitate is lyophilized and reconstituted, and the immunohistochemical staining performance does not change.

[0048] The lyophilized powder of the quality control product of the application has a prolonged storage time, is convenient for transportation, has good dispersibility of the cell suspension after reconstitution, and has normal immunohistochemical staining performance. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 shows the cell dispersion after reconstitution of the lyophilized powder of the liquid SK-OV-3 cell quality control product prepared by using different lyophilization protective solutions, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyophilization protective solution #1; D: reconstitution after lyophilization with lyophilization protective solution #2; E: reconstitution after lyophilization with lyophilization protective solution #3; F: reconstitution after lyophilization with lyophilization protective solution #4; G: reconstitution after lyophilization with lyophilization protective solution #5.

[0050] Figure 2 shows the immunohistochemical staining performance of the corresponding target CK7 after reconstitution of the lyophilized powder of the liquid SK-OV-3 cell quality control product prepared by using different lyophilization protective solutions, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyophilization protective solution #1; D: reconstitution after lyophilization with lyophilization protective solution #2; E: reconstitution after lyophilization with lyophilization protective solution #3; F: reconstitution after lyophilization with lyophilization protective solution #4; G: reconstitution after lyophilization with lyophilization protective solution #5.

[0051] Figure 3 shows the cell dispersion after reconstitution of the lyophilized powder of the liquid Molt-4 cell quality control product prepared by using different solvents, A: cell suspension before lyophilization; B: reconstitution with water; C: reconstitution with 25% alcohol; D: reconstitution with semi-solid buffer.

[0052] Figure 4 shows the immunohistochemical staining performance of the corresponding target CD3 after reconstitution of the lyophilized powder of the liquid Molt-4 cell quality control product prepared by using different solvents, A: cell suspension before lyophilization; B: reconstitution with water; C: reconstitution with 25% alcohol; D: reconstitution with semi-solid buffer.

[0053] Figure 5 shows the cell dispersion after reconstitution of the lyophilized powder of the liquid Molt-4 cell quality control product prepared by using lyophilization protective solution #1 and ddH2O respectively, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyophilization protective solution #1.

[0054] Figure 6 shows the immunohistochemical staining performance of the reconstituted liquid Molt-4 cell quality control lyophilized powder prepared with lyoprotectant #1 and ddH2O respectively for the target point CD3, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyoprotectant #1.

[0055] Figure 7 shows the cell dispersion of the reconstituted liquid Ramos cell quality control lyophilized powder prepared with lyoprotectant #1 and ddH2O respectively, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyoprotectant #1.

[0056] Figure 8 shows the immunohistochemical staining performance of the reconstituted liquid Ramos cell quality control lyophilized powder prepared with lyoprotectant #1 and ddH2O respectively for the target point CD20, A: cell suspension before lyophilization; B: reconstitution after lyophilization with ddH2O; C: reconstitution after lyophilization with lyoprotectant #1. DETAILED DESCRIPTION

[0057] Unless otherwise indicated, all parts and percentages expressed herein are on a weight basis, and all tests and measurements are made in accordance with methods synchronous with the filing date of this application. To the extent that any patent, patent application, or publication is cited in this application, the same is hereby incorporated by reference in its entirety into this application, and the equivalent thereof is intended to be expressly incorporated by reference into this application, particularly with respect to the definitions of terms used therein. If any definition explicitly provided in this application is inconsistent with the definition of a term in the art having priority to the filing date of this application, the definition provided in this application shall control.

[0058] Numerical ranges expressed in the present application as comprising one or more values are approximate ranges and thus include values outside the stated range, unless otherwise indicated. Numerical ranges include all values from and including the lower and the upper values, in increments of one unit, provided that there is a separation of at least two units between any lower value and any higher value. As an example, if a range is stated as 1 to 5, it is intended to encompass, inter alia, the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.2, 2.3, 2.4, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 5.1, 5.2, 5.3, 5.4, and 5.5. These are only examples of what is specifically

[0059] The terms "comprising", "including", "containing", and "having" together with their conjugations do not exclude any other components, steps or processes not expressly stated. For the avoidance of doubt, any composition described herein as comprising, including, containing or having any element can comprise, include, contain or have any additional additional additives, adjuvants or compounds unless expressly stated otherwise. In contrast, the term "consisting essentially of does exclude any component, step or process not expressly stated in the description immediately following such form of words. The term "consisting of does not include any component, step or process not specifically described or listed. The term "or" means any single member of a listed series, or any combination thereof, unless expressly stated otherwise.

[0060] In order to make the technical problems, technical schemes and beneficial effects solved by the present application clearer, the present application will be further described in detail below in combination with embodiments.

[0061] The following examples are presented to demonstrate preferred embodiments of the present application. Those skilled in the art will appreciate that the technology disclosed in the following examples represents the best attempts of the inventors to use the technology discovered by the inventors to practice the present application, and therefore can be considered preferred modes for practicing the present application. However, those skilled in the art will appreciate from the present disclosure that the particular examples disclosed herein can be modified in many ways without departing from the spirit or scope of the present application.

[0062] Unless otherwise defined, 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 application belongs. The materials disclosed herein are cited for their disclosure prior to the filing date of the present application. The citation of a reference herewith is made solely for purposed of presenting the information contained therein.

[0063] Those skilled in the art will appreciate that many of the specific embodiments of the present application described herein can be made without departing from the spirit or scope of the present application. Many modifications will be readily apparent to those skilled in the art, such as can result from the substitution of equivalent techniques for those described or from the use of equivalent technical materials which are known in the art. Such modifications are intended to fall within the scope of the claims.

[0064] The experimental methods in the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.

[0065] Example 1 Liquid cytoplasmic quality control product freeze-drying protective solution

[0066] A freeze-drying protective solution #1 for preparing a freeze-dried powder of a liquid cytoplasmic quality control product is obtained by adding 2% sucrose, 3% glucose, 1% Tween-80 and 0.5% glycine to ddH2O as a solvent.

[0067] To demonstrate the excellent effect of the above freeze-drying protective solution, the inventors also prepared a freeze-drying protective solution with the following formula.

[0068] Freeze-drying protective solution #2: ddH2O as solvent, add 5% sucrose, 1% Tween-80 and 0.5% glycine;

[0069] Freeze-drying protective solution #3: ddH2O as solvent, add 2% sucrose, 3% trehalose, 1% Tween-80 and 0.5% glycine.

[0070] Freeze-drying protective solution #4: ddH2O as solvent, add 4% sucrose, 1% glucose, 1% Tween-80 and 0.5% glycine.

[0071] Freeze-drying protective solution #5: ddH2O as solvent, add 1% sucrose, 4% glucose, 1% Tween-80 and 0.5% glycine.

[0072] Example 2 Preparation process of liquid cytoplasmic control article freeze-dried powder

[0073] 1. Cell suspension preparation

[0074] The cell suspension was prepared according to the method of Example 1 in Chinese invention patent CN112611614B - "Preparation method of cell semi-solid suspension and application thereof":

[0075] The wax-embedded cell sample was sectioned, and the thin section was placed in a centrifuge tube. About 25 mL of deparaffinizing solution was poured into the centrifuge tube and shaken to dissolve the paraffin in the section. The mixture was centrifuged at 2500 rpm for 5 min, and the deparaffinizing solution was poured out. After adding 25 mL of isopropanol, the cell precipitate was shaken with ethanol. The mixture was centrifuged at 2500 rpm for 5 min, and the isopropanol was poured out. About 1 mL of anhydrous ethanol was added to the centrifuge tube and evenly blown with a pipette tip. The mixture was placed in a 1.5 mL centrifuge tube and centrifuged at 2500 rpm for 5 min, and the ethanol was aspirated with a pipette tip.

[0076] 1 mL of freeze-drying protective solution was added to the cell precipitate containing 2.5 M-4.2 M cell fragments to obtain the cell suspension to be freeze-dried.

[0077] 2. Dispensing

[0078] The cell suspension obtained in step 1 was dispensed into freeze-drying containers, with 0.3 mL as one portion.

[0079] 3. Quick freezing

[0080] The cell suspension after dispensing in step 2 was placed in a low-temperature environment below -80°C together with the freeze-drying container for quick freezing;

[0081] 4. Freeze-drying

[0082] First, the in-situ freeze dryer (Mercury 2.5L, Hangzhou Fuerjie Technology Co., Ltd.) was pre-cooled to below -50℃, and the solid obtained by quick freezing in step 3 was warmed together with the freeze-drying container, and the drying was divided into 6 stages:

[0083] (7) The vacuum degree was controlled at 100Pa-150Pa, and the temperature was raised from -80℃ to -20℃, and the temperature was kept for 2h;

[0084] (8) Vacuum pumping; the vacuum degree was reduced to 0.04mBar within 5min, and the temperature was reduced from -20℃ to -50℃, and the temperature was kept for 3h;

[0085] (9) The temperature was raised from -50℃ to -30℃, and the temperature was kept for 5h;

[0086] (10) The temperature was raised from -30℃ to -20℃, and the temperature was kept for 5h;

[0087] (11) The temperature was raised from -20℃ to 0℃, and the temperature was kept for 3h;

[0088] (12) The temperature was raised from 0℃ to 25℃, and the temperature was kept for 6h, and the freeze-drying process was completed, and the freeze-dried powder of the freeze-dried product was formed.

[0089] 5. Storage

[0090] The freeze-dried powder of the cell cytosol control harvested in step 4 was sealed and stored, and packaged.

[0091] Example 3 Performance of freeze-dried powder of cell cytosol control prepared by different freeze-drying protective solutions

[0092] Using the freeze-drying protective solutions #1-#5 in Example 1 and ddH2O (without adding protective components), the liquid SK-OV-3 cell cytosol control was freeze-dried according to the method of Example 2, and different SK-OV-3 cell cytosol control freeze-dried powders were obtained.

[0093] After the SK-OV-3 cell cytosol control freeze-dried powder prepared by different freeze-drying protective solutions was redissolved with 25% alcohol, it was spotted using the automatic slide processing system Plus60 of HUOLING (Hangzhou) Biomedicals Co., Ltd., dried, and then subjected to immunohistochemical experiment to verify the staining performance, and the liquid SK-OV-3 cell cytosol control before freeze-drying was used as a control.

[0094] Immunohistochemical method: The experiment was performed using the LYNX480 automatic immunohistochemical stainer of HUOLING (Hangzhou) Biomedicals Co., Ltd., and the target primary antibody, secondary antibody, and auxiliary reagents used were purchased from the company's commercial kit. The experiment adopted the conventional immunohistochemical experimental process according to the instructions of the kit.

[0095] The SK-OV-3 cell quality control product freeze-dried powder prepared by using the freeze-drying protective solution was reconstituted, and then hematoxylin staining and blue returning were performed to observe the cell dispersion, and the results are shown in Figure 1. As can be seen from Figure 1, when ddH2O (without adding protective components) is used as the solvent for freeze-drying, the cell fragments are stacked after reconstitution, the dispersion is uneven, the density is significantly reduced, and there is loss; when a single sugar is added (freeze-drying protective solution #2) and glucose is replaced by trehalose (freeze-drying protective solution #3), the cell fragments are stacked in a small amount after reconstitution, and the dispersion is slightly uneven; when the rest of the formulations (freeze-drying protective solution #1, #4, #5) are used for freeze-drying, the cell fragments are rarely stacked after reconstitution, and the dispersion is uniform.

[0096] The results of the performance verification of the immunohistochemical staining of SK-OV-3 cells corresponding to the target point CK7 are shown in Figure 2. As can be seen, when ddH2O (without adding protective components) is used as the solvent for freeze-drying, the immunohistochemical staining is not clear due to cell stacking; when a single sugar is added (freeze-drying protective solution #2) and glucose is replaced by trehalose (freeze-drying protective solution #3), the staining performance is slightly worse; and when the rest of the formulations (freeze-drying protective solution #1, #4, #5) are used for freeze-drying, the staining performance is basically the same as before freeze-drying.

[0097] The above results all show that: the SK-OV-3 cell quality control product freeze-dried powder prepared by using freeze-drying protective solution #1, #4, #5 has uniform cell dispersion and good staining performance, and the staining effect of the SK-OV-3 cell quality control product freeze-dried powder prepared by using freeze-drying protective solution #1 is the best after reconstitution.

[0098] The inventors further replaced the surfactant Tween-80 with Tween-20 or replaced the buffer and filler glycine with histidine or lysine which also have the functions of buffer and filler based on freeze-drying protective solution #1, and found that the quality control product freeze-dried powder prepared by using the freeze-drying protective solution has very uniform cell dispersion after reconstitution, and the staining performance does not decrease significantly.

[0099] The inventors also found that the cell dispersion effect and staining performance are not affected when the concentration of sucrose is in the range of 1-4% (i.e. the ratio of sucrose and glucose is 1:4-4:1), the concentration of surfactant (Tween-80, Tween-20, polyethylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, Span-80, Span-20 or poloxamer) is in the range of 0.5-1%, and the concentration of buffer (glycine, histidine or lysine) is in the range of 0.3-0.5%.

[0100] Example 4 Cell quality control product freeze-dried powder prepared by using a conventional freeze-drying process

[0101] To verify the effect of the gradient freeze-drying process of Example 2, the inventors used the freeze-drying protective solutions #1-#5 in Example 1 and ddH2O (without adding protective components) to perform freeze-drying operation on the liquid SK-OV-3 cell quality control product according to the conventional freeze-drying method.

[0102] The conventional freeze-drying method is as follows:

[0103] The cell suspension was rapidly frozen in a -80°C environment, and the freeze-drying container was placed in a pre-cooled freeze-dryer. The partition was not heated or kept warm, and the vacuum operation was directly performed at room temperature. The vacuum degree was reduced to 0.04 mBar within 5 min, and the vacuum state was maintained for 36 h to complete the freeze-drying operation.

[0104] It was found that no matter which freeze-drying protective solution was used, the freeze-dried powder after reconstitution had cells stacked together and could not be dispersed for detection. This indicates that the freeze-drying protective solution of the application needs to be used in cooperation with the freeze-drying process of Example 2 to obtain a quality control product freeze-dried powder with excellent performance.

[0105] Example 5: Effect of different reconstitution solvents on detection results

[0106] The freeze-drying operation was performed on the liquid Molt-4 cell quality control product according to the method of Example 2 using the freeze-drying protective solution #1 in Example 1 to obtain a Molt-4 cell quality control product freeze-dried powder.

[0107] The Molt-4 cell quality control product freeze-dried powder was reconstituted using the following three solvents:

[0108] (1) water;

[0109] (2) 25% alcohol;

[0110] (3) the semi-solid buffer in CN112611614B: including a water-retaining agent and a preservative, the water-retaining agent being 10% by volume polyethylene glycol, the preservative being 1% by volume arginine and 0.01% by volume sodium azide, and further including 0.1% by volume eosin solution.

[0111] After the Molt-4 cell quality control product freeze-dried powder was reconstituted using different solvents, hematoxylin staining and blue returning were first performed to observe the cell dispersion, and the results are shown in FIG. 3. As can be seen from FIG. 3, the cell fragments were uniformly dispersed after reconstitution with different solvents, and were basically the same as before freeze-drying.

[0112] The immunohistochemical staining performance verification results of the Molt-4 cell corresponding target CD3 are shown in FIG. 4. As can be seen, the immunohistochemical staining performance was good after reconstitution with different solvents, and was basically the same as before freeze-drying.

[0113] In summary, after using the freeze-drying process of Embodiment 2 of the present application, the cell dispersion and staining performance have little difference no matter which solvent is used for reconstitution, and meet the requirements of detection.

[0114] Example 6 Freeze-drying effect of freeze-drying protective solution #1 on liquid Molt-4 cell quality control

[0115] The liquid Molt-4 cell quality control was freeze-dried by using the freeze-drying protective solution #1 in Embodiment 1 and ddH2O (without adding protective components) according to the method of Embodiment 2 to obtain Molt-4 cell quality control freeze-dried powder.

[0116] After the Molt-4 cell quality control freeze-dried powder was reconstituted by using 25% alcohol, hematoxylin staining and blue returning were first used to observe the cell dispersion, and the results were compared with the cell suspension before freeze-drying, as shown in FIG. 5. As can be seen from FIG. 5, when ddH2O (without adding protective components) is used as the solvent for freeze-drying, the cell fragments are stacked, dispersed unevenly, and the density is significantly reduced, and there is loss; when the freeze-drying protective solution #1 is used for freeze-drying, the cell fragments are rarely stacked and are evenly dispersed.

[0117] The results of the immunohistochemical staining performance verification of Molt-4 cells corresponding to the target point CD3 are shown in FIG. 6, and it can be seen that when ddH2O (without adding protective components) is used as the solvent for freeze-drying, the immunohistochemical detection staining cannot be seen due to cell stacking; when the freeze-drying protective solution #1 is used for freeze-drying, the staining performance is basically the same as before freeze-drying.

[0118] In summary, after using the freeze-drying protective solution #1 in Embodiment 1 to freeze-dry the liquid Molt-4 cell quality control, the uniformity of the cell fragments and the staining performance of the Molt-4 cell quality control freeze-dried powder after reconstitution are basically the same as before freeze-drying; relatively, after using ddH2O to freeze-dry the liquid Molt-4 cell quality control, the cell fragments after reconstitution of the Molt-4 cell quality control freeze-dried powder are uneven, and the staining performance is poor.

[0119] Example 7 Freeze-drying effect of freeze-drying protective solution #1 on liquid Ramos cell quality control

[0120] The liquid Ramos cell quality control was freeze-dried by using the freeze-drying protective solution #1 in Embodiment 1 and ddH2O (without adding protective components) according to the method of Embodiment 2 to obtain Ramos cell quality control freeze-dried powder.

[0121] The Ramos cell quality control lyophilized powder was reconstituted with 25% alcohol, and then hematoxylin staining and blue returning were used to observe the cell dispersion, and the results were compared with the cell suspension before lyophilization, as shown in Figure 7. As can be seen from Figure 7, the cell fragments were stacked, unevenly dispersed, and the density was significantly reduced, and there was loss when ddH2O (without adding protective components) was used as the solvent for lyophilization; the cell fragments were rarely stacked and evenly dispersed when the lyophilization protective solution #1 was used for lyophilization.

[0122] The results of the performance verification of immunohistochemical staining of Ramos cells corresponding to the target point CD20 are shown in Figure 8. As can be seen, the immunohistochemical detection staining was not clear due to cell stacking when ddH2O (without adding protective components) was used as the solvent for lyophilization; the staining performance was basically the same as before lyophilization when the lyophilization protective solution #1 was used for lyophilization.

[0123] In summary, the lyophilization of the liquid Ramos cell quality control with the lyophilization protective solution #1 in Example 1 resulted in a lyophilized powder of the Ramos cell quality control, which was basically the same in terms of cell fragment uniformity and staining performance after reconstitution as before lyophilization. In contrast, the lyophilization of the liquid Ramos cell quality control with ddH2O resulted in a lyophilized powder of the Ramos cell quality control, which was not uniform in terms of cell fragments and had poor staining performance after reconstitution.

[0124] Example 8 Lyophilization recovery rate experiment of liquid Molt-4 cell quality control

[0125] The liquid Molt-4 cell quality control was used for lyophilization recovery rate verification. 4.0 mL of qualified liquid Molt-4 cell quality control was taken, solvent exchanged, and then divided into 8 equal parts. Lyophilization protective solution #1 and ddH2O (without adding protective components) were used for lyophilization of 4 parts each. After pre-freezing at -80℃, lyophilization was performed. The process used the conditions and parameters of Example 2. After lyophilization, 8 bottles of Molt-4 cell quality control lyophilized powder were obtained.

[0126] After reconstitution with 500 μL of 25% alcohol, flow cytometry (MAC & Quant Analyzer) was used for counting. 50 μL of the reconstituted solution was centrifuged. The supernatant was discarded, 1 mL of 1xPBS was added for resuspension, and the solution was diluted 20 times. 20 μL of the diluted solution was taken for FACS sample loading. The process was repeated until all samples were tested. The number of Molt-4 cell fragments in 20 μL of the sample was A. The number of Molt-4 cell fragments before lyophilization was B. The lyophilization recovery rate was calculated using the following formula.

[0127] Recovery rate% = A / B

[0128] The results are shown in Table 1.

[0129] Table 1 Lyophilization recovery rate results of liquid Molt-4 cell quality control

[0130]

[0131] Note: Control is the cell count result of liquid Molt-4 cell quality control before freeze-drying.

[0132] As shown in Table 1, the recovery rate of the liquid Molt-4 cell quality control using freeze-drying protective solution #1 is more than 70%, and the highest is more than 80%; while the recovery rate of the liquid Molt-4 cell quality control using ddH2O is only 35%-40%.

[0133] Example 9 Freeze-drying recovery rate experiment of liquid Ramos cell quality control

[0134] The freeze-drying recovery rate of the liquid Ramos cell quality control was verified. 4.0 mL of qualified liquid Ramos cell quality control was taken, and after solvent replacement, it was divided into 8 parts. Freeze-drying was performed using freeze-drying protective solution #1 and ddH2O (without adding protective components), and after pre-freezing at -80°C, freeze-drying was performed. The process used the conditions and parameters of Example 2. After freeze-drying, 8 bottles of Ramos cell quality control freeze-dried powder were obtained.

[0135] After being fully reconstituted with 500 μL of 25% alcohol, the same counting method of Example 7 was used to count and calculate the freeze-drying recovery rate, and the results are shown in Table 2.

[0136] Table 2 Freeze-drying recovery rate results of liquid Ramos cell quality control

[0137]

[0138] Note: Control is the cell count result of liquid Ramos cell quality control before freeze-drying.

[0139] As shown in Table 1, the recovery rate of the liquid Ramos cell quality control using freeze-drying protective solution #1 is more than 70%, and the highest is more than 80%; while the recovery rate of the liquid Ramos cell quality control using ddH2O is only 30%-40%.

[0140] All documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications can be made to this application by those skilled in the art, and such changes and modifications are intended to fall within the scope of the claims accompanying this application.

Claims

1. A quality control lyophilized powder, characterized in that, The quality control sample freeze-dried powder is obtained by freeze-drying a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifugation after slicing, deparaffinizing and hydrating a paraffin-embedded cell sample for immunohistochemical detection, The cell precipitate is freeze-dried in a freeze-drying protective solution, wherein the freeze-drying protective solution comprises excipients and a surfactant, the excipients comprise stabilizers, fillers and buffers, The stabilizers comprise sucrose and glucose, and the ratio of sucrose to glucose is 1:4 to 4:

1.

2. The quality control lyophilized powder of claim 1, wherein, The fillers are at least one selected from the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and derivatives thereof; and the buffers are at least one selected from the group consisting of phosphate, histidine, glycine, lysine, citrate.

3. The quality control lyophilized 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. The quality control lyophilized powder according to claim 1 or 2, characterized in that, The freeze-drying is gradient freeze-drying at -80°C to 0°C.

5. The quality control lyophilized powder of claim 4, wherein, The gradient freeze-drying at -80°C to 0°C is specifically: (1) the vacuum degree is 100 Pa to 150 Pa, and the temperature is raised from about -80°C to about -20°C, and the temperature is kept for 1.5 to 2.5 h; (2) the vacuum degree is 0.02 to 0.05 mBar, and the temperature is lowered from about -20°C to about -50°C, and the temperature is kept for 2.5 to 3.5 h; (3) the temperature is raised from about -50°C to about -30°C, and the temperature is kept for 4.5 to 5.5 h; (4) the temperature is raised from about -30°C to about -20°C, and the temperature is kept for 4.5 to 5.5 h; (5) the temperature is raised from about -20°C to about 0°C, and the temperature is kept for 2.5 to 3.5 h; (6) the temperature is raised from about 0°C to about 25°C, and the temperature is kept for 5.5 to 6.5 h.

6. Use of a lyoprotectant solution for the preparation of a lyophilized powder of a cell pellet, characterized in that, The cell precipitate contains cell fragments, is obtained by centrifugation after slicing, deparaffinizing and hydrating a paraffin-embedded cell sample for immunohistochemical detection, the freeze-drying protective solution comprises excipients and a surfactant, the excipients comprise stabilizers, fillers and buffers, the stabilizers comprise sucrose and glucose, and the ratio of sucrose to glucose is 1:4 to 4:1; the fillers are at least one selected from the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and derivatives thereof; the buffers are at least one selected from the group consisting of phosphate, histidine, glycine, lysine, citrate, and the cell precipitate is freeze-dried in the freeze-drying protective solution.

7. Use according to claim 6, 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.

8. Use according to claim 7, characterized in that, The freeze-drying protective solution comprises 3% to 8% stabilizers, 0.5% to 1.5% Tween-80 and 0.3% to 0.5% glycine.

9. A cell suspension, characterized in that, The cell suspension is obtained by mixing a freeze-drying protective solution and a cell precipitate containing cell fragments, wherein the cell precipitate is obtained by centrifugation after slicing, deparaffinization and hydration treatment of a paraffin-embedded cell sample for immunohistochemical detection, the freeze-drying protective solution comprises an excipient and a surfactant, the excipient comprises a stabilizer, a filler and a buffer, the stabilizer comprises sucrose and glucose, the ratio of sucrose and glucose is 1:4~4:1; the filler is at least one selected from the group consisting of sucrose, lactose, histidine, glycine, lysine, mannitol, sorbitol, microcrystalline cellulose, starch and derivatives thereof; the buffer is at least one selected from the group consisting of phosphate, histidine, glycine, lysine, citrate, and 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.

10. A cell suspension, characterized in that, The quality control product freeze-dried powder according to any one of claims 1~5 is reconstituted by using a solvent.

11. The method of preparing a quality control lyophilized powder according to claim 1, characterized in that, The method further comprises a step of freeze-drying the cell precipitate in the freeze-drying protective solution.

12. Use of the lyophilized powder according to any one of claims 1 to 5 for the preparation of a kit for immunoassay, characterized in that, The immunodetection is selected from one of the group consisting of immunohistochemistry, immunocytochemistry and in situ hybridization.

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