Method for detecting proliferation capacity of cells in three-dimensional porous microcarrier material

Abstract

The invention belongs to the technical field of biological cell visual detection, and particularly relates to a method for detecting the proliferation capacity of cells in a three-dimensional porous microcarrier material, which comprises the following steps: respectively preparing a cell suspension and a porous microcarrier material filled with a complete culture medium solution, uniformly mixing and culturing; digesting the microcarrier material; cleaning, filtering and collecting the microcarrier material gathered above the screen; preparing a low-melting-point agarose solution, uniformly mixing the low-melting-point agarose solution with the complete culture medium solution to serve as a bottom culture plate / observing and monitoring the cooling temperature of the solution; uniformly mixing a low-melting-point agarose solution with the cooling temperature of 38 + / -2 DEG C and a microcarrier material, and inversely placing above the bottom culture plate to form a middle layer; adding a complete culture medium solution to the upper surface of the middle layer to form a liquid nutrient moisturizing layer; placing the culture material in an incubator, and standing and culturing; and the proliferation activity of the cells in the microcarrier material is judged by calculating the formation rate of the microcarrier cell colonies.

Description

Technical Field

[0001] This invention belongs to the field of biological cell visualization detection technology, specifically relating to a method for detecting the cell proliferation capacity inside a three-dimensional porous microcarrier material. Background Technology

[0002] Porous microcarrier materials are core scaffold materials in the field of three-dimensional cell culture. With their high specific surface area, biomimetic porous structure and good cell compatibility, they have become key carriers connecting two-dimensional planar culture and complex organ construction. By simulating the three-dimensional microenvironment in vivo, porous microcarriers further break through the limitations of two-dimensional culture and solid carriers, providing cells with a three-dimensional space for proliferation, differentiation and migration. They have irreplaceable application value in large-scale cell preparation, tissue engineering and drug development.

[0003] However, it has significant limitations in visually monitoring cell growth status: while surface cell growth can be directly observed using conventional microscopy, the growth dynamics of internal cells are difficult to characterize precisely. Observation of cells inside microcarriers is constrained by multiple factors, including: 1) The optical properties of the material itself constitute a physical barrier: Most porous microcarrier materials exhibit some optical opacity or scattering effects, especially those with particle sizes greater than 100 μm and porosity less than 80%. Visible light or fluorescence cannot effectively penetrate to the core region, resulting in the masking of internal cell signals; 2) Probe transmission and signal attenuation are prominent issues: Fluorescent staining probes (such as live cell dyes and antibody probes) need to diffuse through the pore network to the interior. However, due to the influence of pore size (diffusion resistance increases significantly when less than 50 μm), pore connectivity, and the hydrophilicity / hydrophobicity of the carrier surface, probes tend to accumulate on the surface, resulting in uneven or even no staining of internal cells, even if stained. 1) The fluorescence signal is successfully transmitted, but it is severely attenuated when penetrating the carrier matrix, making it difficult for the detector to capture it effectively; 2) The penetration capability of existing microscopy techniques is limited: the effective penetration depth of ordinary inverted fluorescence microscopes is only 20-50 μm, which cannot cover the internal area of ​​large-diameter microcarriers; although confocal laser scanning microscopes can achieve three-dimensional reconstruction through layer-by-layer scanning, the resolution of the core area drops significantly for carriers with a particle size of more than 200 μm, and the detection time is long, making it difficult to meet the needs of rapid screening of batch samples; while two-photon microscopes have a deeper penetration depth (up to 500 μm) and can distinguish between live and dead cells through autofluorescence without fluorescent probes, they are limited by expensive equipment and low adoption rate.

[0004] The aforementioned limitations directly affect the completeness and accuracy of experimental results. Researchers may misjudge the overall culture effect based solely on the good condition of surface cells, neglecting internal problems such as cell hypoxia and necrosis, proliferation arrest, or abnormal differentiation caused by insufficient nutrient diffusion and accumulation of metabolic waste. In large-scale cell preparation, tissue engineering, and drug development, the proliferation capacity and state of internal cells are closely related to cell yield, overall tissue activity, and drug responsiveness. This "visible on the surface, unknown inside" monitoring blind spot may lead to distorted evaluation results of biocompatibility of different types of porous carrier materials and biases in research on cell culture systems based on porous microcarrier materials; thus becoming a key technical bottleneck restricting the further promotion and application of porous microcarrier materials.

[0005] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0006] This disclosure provides at least one method for detecting the cell proliferation capacity inside a three-dimensional porous microcarrier material.

[0007] In a first aspect, embodiments of this disclosure provide a method for detecting the cell proliferation capacity within a three-dimensional porous microcarrier material, comprising the following steps: S1, preparing a cell suspension and a porous microcarrier material with its internal space filled with a complete culture medium solution, mixing them, and then inoculating them into an ultra-low adhesion cell culture plate for static culture, periodically changing the complete culture medium solution; S2, observing the binding and growth of cells with the porous microcarrier material using a microscope, stopping the culture when the surface of the microcarrier material is observed to be coated with cells, and collecting the porous microcarrier material with surface-coated cells enriched above the sieve using a cell sieve; S3, digesting the microcarrier material obtained in S2 with a mild trypsin, observing the cell shedding on the surface of the microcarrier material by digestion and microscopic examination, stopping digestion when all surface cells have detached; S4, washing the digested microcarrier material in S3 with DPBS phosphate buffer, and collecting the microcarrier material aggregated above the sieve using a cell sieve; S5, preparing a 0.8%-1.5% low-melting-point agarose solution with distilled water, sterilizing it under high temperature and high pressure, and taking... Sterilized agarose solution and complete culture medium solution were mixed at a volume ratio of 1:1. The mixed solution was then inverted in a culture vessel to cool and fix, serving as the bottom culture plate. S6: A 0.3%-0.6% low-melting-point agarose solution was prepared using distilled water and autoclaved. The sterilized agarose solution was then mixed with the complete culture medium solution at a volume ratio of 1:1 and allowed to stand. The cooling temperature of the solution was observed and monitored. S7: The low-melting-point agarose solution cooled to 38℃±2℃ from S6 and the microcarrier material obtained in step S4 were thoroughly mixed. After homogenization, invert the plate above the bottom culture plate and allow it to cool and solidify to form an intermediate layer; S8, add complete culture medium solution to the upper surface of the intermediate layer of the culture vessel to form a liquid nutrient and moisture-retaining layer covering the upper surface, thus obtaining the culture body; S9, place the culture body prepared in step S8 in an incubator and incubate statically for 5-10 days. Stop the culture when visible cell clusters are observed around the microcarrier material; S10, use the visible cell clusters as the standard for counting colonies and determine the cell proliferation activity inside the microcarrier material by calculating the microcarrier cell colony formation rate.

[0008] In one optional embodiment, the cell suspension in S1 is prepared by selecting animal adherent cells that are in good growth condition and in the logarithmic growth phase.

[0009] In one alternative embodiment, the animal adherent cells in S1 include mouse fibroblast L-929 cells.

[0010] In one optional embodiment, the method for preparing porous microcarrier material in S1 with its internal space filled with complete culture medium solution includes: placing the porous microcarrier material in a container containing complete culture medium solution, and using a vacuum device to fill the gaps in the microcarrier material with complete culture medium solution until the microcarrier material completely sinks to the bottom of the container.

[0011] In one alternative embodiment, the volume of the porous microcarrier material in the container of S1 does not exceed 1 / 3 of the volume of the complete culture medium solution.

[0012] In one optional embodiment, the ratio of cell seeding amount to microcarrier material in the cell suspension in S1 is: 100,000 cells / 1 mg porous microcarrier material.

[0013] In one optional embodiment, the pore size of the cell sieve in S2 is larger than the diameter of the porous microcarrier material, but smaller than the diameter of the porous microcarrier material on which the cells are wrapped.

[0014] In one optional embodiment, the washing in S4 is performed with DPBS phosphate buffer at least 3 times; the pore size of the cell sieve in S4 is smaller than the diameter of the porous microcarrier material.

[0015] In an optional implementation, the microcarrier cell colony formation rate (%) in S10 is calculated as: (number of colonies / number of microcarriers inoculated) × 100%.

[0016] In one alternative embodiment, the complete culture medium comprises 2% penicillin-streptomycin antibiotic and 20% fetal bovine serum by volume percentage.

[0017] The method for detecting the cell proliferation capacity inside this three-dimensional porous microcarrier material has the following advantages: 1. By co-culturing porous microcarrier materials filled with different proportions of seed cells and complete culture medium in low-adhesion culture dishes, porous microcarrier materials loaded with cells can be easily obtained. Compared with existing technologies, there is no need to use large shake flask culture devices, and the operation is simpler. 2. By utilizing multiple digestions and cell sieving with mild trypsin, porous microcarrier materials with no obvious cell load on the surface are obtained while better maintaining the viability of cells inside the microcarriers. Compared with existing technologies, it is not necessary to obtain the cells inside the microcarrier materials by destroying their structure, thus maintaining the structural integrity of the microcarrier materials themselves. 3. The soft agar colonization method using porous microcarrier materials allows for a direct assessment of the proliferation capacity of cells within the microcarrier by observing the number and size of colonies. This avoids the complexity of existing fluorescent probe staining techniques such as live / dead staining and immunofluorescence staining, as well as the limitations of fluorescent dyes and probes in reaching the interior of the microcarrier material.

[0018] 4. Compared with the prior art, the technical solution of the present invention can efficiently detect the proliferation activity of cells inside multiple different types of porous microcarriers in the same batch, and can also detect the growth status of cells inside the same type of porous microcarrier materials in different cell culture systems.

[0019] 5. The technical solution in this invention is simple to operate, and the required reagents and consumables are all common and conventional reagents and consumables. The instruments and equipment used are all conventional instruments and equipment. It eliminates the defects of complexity and low universality in the use of large-scale instruments and equipment such as laser confocal microscopes and two-photon microscopes, and also avoids the use of high-priced reagents such as fluorescent probes, which greatly saves detection costs.

[0020] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of a vacuuming method for a porous microcarrier material provided in an embodiment of this disclosure; Figure 2 A schematic diagram illustrating the co-culture of porous microcarrier materials and cells according to an embodiment of this disclosure; Figure 3 A schematic diagram of a porous microcarrier material before and after mild trypsin digestion, provided in an embodiment of this disclosure; Figure 4 A schematic diagram (multilayer agar plate schematic diagram) of a porous microcarrier colonized in soft agar according to an embodiment of this disclosure. Figure 5 This is a schematic diagram illustrating the continuous culture of cells inside a porous microcarrier material on soft agar, as provided in an embodiment of this disclosure. Figure 6 This is a schematic diagram of porous microcarrier colonies on soft agar provided in an embodiment of this disclosure. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] As used herein, the phrases “in one embodiment,” “according to one embodiment,” “in some embodiments,” etc., generally refer to the fact that a particular feature, structure, or characteristic following the phrase can be included in at least one embodiment of this disclosure. Therefore, a particular feature, structure, or characteristic can be included in more than one embodiment of this disclosure, such that these phrases do not necessarily refer to the same embodiment. As used herein, the terms “example,” “exemplary,” etc., are used to “serve as an example, instance, or illustration.” Any implementation, aspect, or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or superior to other implementations, aspects, or designs. Rather, the use of the terms “example,” “exemplary,” etc., is intended to present concepts in a specific manner.

[0026] The terminology used herein is for the purpose of describing specific exemplary configurations only and is not intended to be limiting. As used herein, the singular articles “a,” “an,” and “the” may also be intended to include plural forms unless otherwise clearly stated herein. The terms “comprising,” “including,” and “having” are inclusive and thus specify the presence of features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein should not be construed as requiring them to be performed in the specific order discussed or shown, unless specifically identified as such. Additional or alternative steps may be employed.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] The cells used in the following examples are L-929 mouse fibroblasts; The porous microcarrier is a porous microcarrier material prepared by Changzhou Institute of Pharmaceutical Research Co., Ltd. Some embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0030] Example 1: A method for co-culturing cells with porous microcarrier materials The specific steps are as follows: 1. Preparation of microcarrier suspension: Weigh 40 mg of porous microcarrier powder after high-temperature and high-pressure sterilization, and place it in 40 mL of RPMI 1640 complete culture medium solution (e.g., Figure 1-1 (As shown), mix well and place in a 50mL syringe (as shown). Figure 1-2 As shown), at this point, due to the presence of internal pores, a portion of the porous microcarrier material floats on the liquid surface. Typically, the volume of the porous microcarrier material in the syringe does not exceed 1 / 3 of the total culture medium solution volume. After sealing the top of the syringe, pump it back and forth (20-30 times). After completing the vacuuming process, the porous microcarrier material sinks to the bottom of the tube (as shown). Figure 1-3 (as shown in 1-4) 2. Cell preparation: Mouse fibroblast L-929 cells in good growth condition and in the logarithmic growth phase were digested with trypsin and counted to prepare a cell suspension; 3. Cell Seeding and Adsorption: The cell suspension and porous microcarrier material were mixed and seeded into 6-well ultra-low adhesion cell culture plates. The ultra-low adhesion culture plates used in this step were purchased from Beyotime Biotechnology Co., Ltd. The seeding ratio was 500,000 fibroblasts: 5 mg of porous microcarrier material. After mixing and seeding, the cells were placed in a cell culture incubator for static culture at 37°C, 5% CO2, 95% air, and saturated humidity for 3-7 days. Complete culture medium solution was added periodically during the culture process. After three days of total culture... Figure 2 As shown, a large number of fibroblasts have been adsorbed on the surface of the porous microcarrier.

[0031] Example 2: A method for screening and collecting porous microcarriers containing cells. The specific steps are as follows: 1. Collection of porous microcarriers adsorbing cells: A certain volume of cell spheres containing porous microcarrier material obtained in Example 1 is collected; in the above step, a cell sieve of a certain size is used to screen and filter the porous microcarrier material enriched on the surface of the sieve and encapsulating cells. The pore size of the cell sieve is larger than the diameter of the porous microcarrier material and smaller than the diameter of the porous microcarrier material encapsulating cells on the surface. 2. Removal of cells from the surface of porous microcarrier materials: The porous microcarriers with adsorbed cells are subjected to multiple digestion treatments with mild trypsin, and the digestion time is determined by observing the digestion of cells on the surface of the porous microcarrier materials. The trypsin used in this step is a mild digestive trypsin, and the shedding of cells from the surface of the porous microcarriers is further determined by microscopy. 3. Collection of cell-free porous microcarrier material: Collect the microcarrier material aggregated above the sieve by rinsing with a cell sieve and DPBS phosphate digestion solution at least three times. This step removes cell residue from the surface and shallow pores of the material, ultimately obtaining porous microcarrier material with no cell adhesion on the carrier surface. The pore size of the selected cell sieve is smaller than the diameter of the porous microcarrier material. Figure 3 As shown, after multiple pancreatic enzyme digestion and sieving, a batch of porous microcarrier materials with almost no cell adsorption on the surface were obtained.

[0032] Example 3: A method for preparing a multilayer agar plate for cell culture. The specific steps are as follows: 1. Prepare 0.8%-1.5% low melting point agarose solutions using distilled water. After high temperature and high pressure sterilization, mix 1% agarose and complete culture medium solution (containing 2% penicillin-streptomycin antibiotics and 20% fetal bovine serum) in a 1:1 ratio. Add 1.5 mL of the mixture to a 6-well plate and let it cool and solidify. This mixture will then be used as the bottom culture plate for later use. 2. Prepare a 0.3%-0.6% low-melting-point agarose solution using distilled water. After autoclaving, mix 0.4% agarose and complete culture medium solution (containing 2% penicillin-streptomycin antibiotics and 20% fetal bovine serum) in a sterile tube at a 1:1 ratio. When the agar gel temperature reaches 38℃±2℃, inject the cell-free microcarrier material collected in Example 2 into the tube. After thorough mixing, place 200-300 μL above the bottom culture plate obtained in Step 1 as an intermediate layer, thereby forming two different agar layers on the upper and lower sides. 3. After the prepared intermediate layer has solidified, add 200-300 μL of complete culture medium solution to form a layer as shown in the image. Figure 4 The multilayer agar culture model shown has an upper nutrient and moisture-retaining layer, a middle soft agar layer with embedded porous microcarrier material, and a bottom hard agar layer.

[0033] Example 4: A method for soft agar colonization culture and proliferation activity detection containing cell-porous microcarrier materials The specific steps are as follows: 1. The culture system obtained in Example 3 was placed in an incubator and statically cultured for 5-10 days. The culture environment was 37°C, 5% CO2, 95% air, and saturated humidity. During the culture period, the culture dish was kept static. The culture was terminated when visible clones appeared around the microcarriers. Figure 5 As shown.

[0034] 2. The presence of visible cell clusters around the microcarrier material was used as the standard for counting colonies, and the colony formation rate was calculated (the average value of the colonies in 3 replicates was used for calculation). Microcarrier cell colony formation rate (%) = number of colonies / number of microcarriers inoculated × 100%. The colony formation rate can be used to determine the proliferation activity of cells within the microcarrier material. In this invention, a colony formation rate of not less than 75% is considered sufficient to determine that the cells within this multi-purpose microcarrier material have good proliferation activity. Figure 6 As shown, the number of porous microcarriers seeded in three wells of the 6-well plate were 45, 37, and 51, respectively; the number of porous microcarriers with visible cell colonies growing around the wells were 40, 31, and 42, respectively; the average colony formation rate of this porous microcarrier material was calculated to be 85%.

[0035] Furthermore, regarding the extension of this embodiment: the comparison of cell proliferation activity within different types of porous microcarrier materials can be determined based on the average colony formation rate; the magnitude of cell proliferation activity within porous microcarrier materials of the same type but different culture methods can be preliminarily determined based on the size of the formed cell colonies and the colony formation rate.

[0036] This invention uses L-929 mouse fibroblasts as an example. Meanwhile, other cells that can be cultured into cell spheroids and microorganisms using ultra-low adhesion cell culture plates are also applicable to the above scheme.

[0037] Furthermore, the methods described in the various embodiments of the present invention can be integrated into one method, or each method can exist independently, or two or more methods can be integrated into one unit.

[0038] In summary, this invention provides a method for detecting cell proliferation activity within porous microcarrier materials through exploration of detection methods. Compared to methods in existing patents, this invention is simple to operate, low in cost, and has a short detection cycle; moreover, it does not require the introduction of large-scale instruments and equipment during the experiment. Furthermore, the method of this invention can more intuitively determine the cell compatibility of different porous microcarrier materials, and is suitable for the application evaluation of various microcarrier materials. At the same time, by collecting cells from the microcarrier material colonies, other biological indicators besides cell proliferation capacity can also be further determined.

[0039] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for detecting the cell proliferation capacity inside a three-dimensional porous microcarrier material, characterized in that, Includes the following steps: S1. Cell suspension and porous microcarrier material with internal space filled with complete culture medium solution were prepared separately. After mixing, they were seeded in ultra-low adhesion cell culture plates and cultured statically. The complete culture medium solution was replaced periodically. S2. Use a microscope to observe the binding and growth of cells with porous microcarrier materials. When it is observed that the surface of the microcarrier material is wrapped with cells, stop the culture and use a cell sieve to filter and collect the porous microcarrier material with cells wrapped on the surface above the sieve. S3: The microcarrier material obtained in S2 was digested using mild trypsin. The cell shedding on the surface of the microcarrier material was observed by microscopic examination during digestion until all surface cells were shed. S4, the digested microcarrier material in S3 was washed with DPBS phosphate buffer and the microcarrier material aggregated above the sieve was collected by filtration through a cell sieve. S5. Prepare a low-melting-point agarose solution with a concentration of 0.8%-1.5% using distilled water, sterilize it under high temperature and high pressure, mix the sterilized agarose solution with the complete culture medium solution at a volume ratio of 1:1, invert the mixed solution into a culture vessel to cool and fix it, and use it as the bottom culture plate. S6. Prepare a low-melting-point agarose solution with a concentration of 0.3%-0.6% using distilled water, sterilize it under high temperature and high pressure, mix the sterilized agarose solution with the complete culture medium solution at a volume ratio of 1:1, let it stand, and observe and monitor the cooling temperature of the solution. S7. Take the low melting point agarose solution cooled to 38℃±2℃ in S6 and the microcarrier material obtained in step S4. Mix the two thoroughly and then invert them on top of the bottom culture plate. After cooling and fixing, an intermediate layer is formed. S8. Add complete culture medium solution to the upper surface of the middle layer of the culture vessel to form a liquid nutrient and moisture-retaining layer covering the upper surface, and obtain the culture body. S9. Place the culture body prepared in step S8 in an incubator and let it stand for 5-10 days. Stop the culture when visible cell clusters are observed around the microcarrier material. S10 uses visible cell clusters as the standard for counting colonies, and judges the cell proliferation activity inside the microcarrier material by calculating the cell colony formation rate of the microcarrier.

2. The detection method as described in claim 1, characterized in that, The cell suspension in S1 is prepared by selecting animal adherent cells that are in good growth condition and in the logarithmic growth phase.

3. The detection method as described in claim 2, characterized in that, The animal adherent cells in S1 include mouse fibroblast L-929 cells.

4. The detection method as described in claim 1, characterized in that, The method for preparing the porous microcarrier material in S1 with its internal space filled with complete culture medium solution includes: placing the porous microcarrier material in a container containing complete culture medium solution, and using a vacuum device to fill the gaps in the microcarrier material with complete culture medium solution until the microcarrier material completely sinks to the bottom of the container.

5. The detection method as described in claim 4, characterized in that, The volume of the porous microcarrier material in the container of S1 does not exceed 1 / 3 of the volume of the complete culture medium solution.

6. The detection method as described in claim 1, characterized in that, The ratio of cell seeding amount to microcarrier material in the S1 cell suspension is 100,000 cells / 1 mg of porous microcarrier material.

7. The detection method as described in claim 1, characterized in that, The pore size of the cell sieve in S2 is larger than the diameter of the porous microcarrier material, but smaller than the diameter of the porous microcarrier material on which the cells are wrapped.

8. The detection method as described in claim 1, characterized in that, The S4 step involves rinsing with DPBS phosphate buffer at least three times. In S4, the pore size of the cell sieve is smaller than the diameter of the porous microcarrier material.

9. The detection method as described in claim 1, characterized in that, The colony formation rate (%) of microcarrier cells in S10 is calculated as: (Number of colonies / Number of microcarriers inoculated) × 100%.

10. The detection method as described in claim 1, characterized in that, The complete culture medium comprises 2% penicillin-streptomycin antibiotics and 20% fetal bovine serum by volume percentage.

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