Method for cultivation of single spheroids of lech-3 cells in a 96-well plate

Abstract

FIELD: biotechnology; medicine.SUBSTANCE: three-dimensional cell cultivation, used to obtain and cultivate single cell spheroids for the purpose of conducting diagnostic studies, testing the safety and effectiveness of preparations, and cultivating viruses. The method involves the formation of single spheroids from the diploid human embryonic lung cell line (LECH-3) in a 96-well plate by creating microwells with a diameter of 500 μm in a two-percent agarose hydrogel, followed by seeding of cells at a density of 20×103 cells / ml. The morphometric parameters of the obtained spheroids are characterized by a high degree of homogeneity with a coefficient of variation of size of 1.18% and roundness of 1.68%.EFFECT: spheroids remain stable and viable for 14 days with regular changes of the nutrient medium, which allows for their long-term use in experimental studies.1 cl, 3 dwg, 2 ex

Description

[0001] Application area

[0002] The invention relates to the field of biotechnology and medicine, in particular to the field of three-dimensional cell cultivation, and can be used to obtain and cultivate single cell spheroids for the purpose of conducting diagnostic studies, testing the safety and efficacy of drugs, culturing viruses, including difficult-to-cultivate ones. The method involves culturing single spheroids of cells from the diploid human embryonic lung cell line (LECH-3) in a 96-well plate by creating microwells with a diameter of 500 μm in a two-percent agarose hydrogel, followed by seeding the cells at a density of 20 × 10³ cells / ml. The morphometric parameters of the obtained spheroids are characterized by a high degree of homogeneity with a coefficient of variation in size of 1.18% and roundness of 1.68%, which is confirmed by statistical analysis using the Spheroanalysis program.Spheroids remain stable and viable for 14 days with regular changes of the nutrient medium, which allows for their long-term use in experimental studies.

[0003] Technology Level

[0004] In the field of three-dimensional cell culture, various approaches have been developed to obtain cell spheroids from fibroblast cultures.

[0005] A method is known for producing three-dimensional structures from human lung fibroblasts, in particular from the MRC-5 cell line, which has the greatest similarity to the LEC-3 line used in the claimed invention.

[0006] The MRC-5 cell line (Medical Research Council cell strain 5) is a diploid fibroblast cell line cultured from the lung tissue of a 14-week-old human male embryo in 1966. This line is widely used in biomedical research and diagnostics, characterized by a normal karyotype and limited proliferative potential (about 50-60 population doublings).

[0007] Rosellini et al. [6] used two methodological approaches to form spheroids from MRC-5 cells. Using the hanging drop method, the authors prepared a cell suspension at a concentration of 500,000 cells / mL and added 20 μL to each well of a 96-well plate. After inverting the plate, the droplets were held in place by surface tension, and the cells accumulated at the liquid-air interface. An alternative method involved culturing on non-adhesive surfaces: the wells of a 6-well plate were coated with poly-2-hydroxyethyl methacrylate (pHEMA), after which 100,000 cells were seeded in 1 mL of DMEM medium with 2% FBS. Spheroids formed after 3-4 days of culturing.

[0008] The authors demonstrated that three-dimensional MRC-5 structures exhibit increased sensitivity to viral infection compared to monolayer cultures, which is due to a more natural cell microenvironment and functional intercellular interactions. However, the described methods have several significant limitations: the hanging drop method is characterized by technical complexity, limited volume of culture medium, and the risk of contamination during plate inversion; culturing on non-adhesive surfaces results in the formation of spheroids with high variability in size and morphology.

[0009] It is important to note that the MRC-5 cell line, despite its advantages, is a foreign development and, given current geopolitical restrictions, presents certain challenges for Russian researchers in terms of availability and cost. Therefore, the development of methods for culturing three-dimensional structures from the domestic LEC-3 cell line, which has similar biological characteristics to MRC-5, is particularly relevant.

[0010] A fundamental step in the development of three-dimensional cell models based on the domestic cell line LECh-3 was the development by the authors of the present invention of a method for culturing herpes simplex virus type 1 (HSV-1) in LECh-3 spheroids formed in a 6-well plate [7]. In this method, LECh-3 spheroids were formed in agarose hydrogel microwells created using a special mold. It was demonstrated that the three-dimensional structures of LECh-3 provide more physiologically relevant conditions for virus replication compared to traditional monolayer cultures.

[0011] However, the method implemented in a 6-well plate has several limitations related to high-throughput screening and automation of the analysis process. The 6-well plate format does not allow for the simultaneous testing of a large number of experimental conditions, which is critical for screening drugs and other biologically active substances. Furthermore, this format requires significant consumption of cell material and reagents, increasing the cost of experiments.

[0012] The objective of the present invention is to develop a method for culturing single spheroids of LEC-3 cells in a 96-well plate, which would provide:

[0013] Fully compatible with standard laboratory equipment used for high-throughput screening.

[0014] High reproducibility and standardization of the parameters of the obtained spheroids, including size, shape and functional characteristics.

[0015] Formation of one spheroid in each well of the plate, which will enable accurate quantitative analysis and increase the reliability of experimental results.

[0016] Efficient use of the domestic cell line LECH-3, which will ensure technological independence from imported materials.

[0017] Economy and labor savings when conducting experiments with a large number of experimental conditions.

[0018] Possibility of long-term cultivation of spheroids while maintaining their viability and functional characteristics.

[0019] Solving this problem will expand the methodological arsenal of domestic research laboratories, increase the efficiency of preclinical studies, and ensure technological independence in the development of new drugs, vaccines, and diagnostic systems. The invention provides: an expanded methodological base for biomedical research, the creation of a standardized platform for high-throughput screening, increased efficiency in preclinical studies of drugs and biological agents, including viruses, improved performance characteristics of 3D cell models, and technological independence in the development of new biomedical technologies through the use of the domestic LEC-3 cell line.

[0020] Technical result: ensuring the formation of single spheroids of LEC-3 cells, homogeneous in size and shape, in the wells of a standard 96-well plate, increasing the reproducibility of experimental data, achieving high standardization of the morphometric parameters of spheroids, increasing the sensitivity of three-dimensional cell culture to biological agents, including herpes simplex virus type 1 (HSV-1 / L2).

[0021] A method for culturing single spheroids of diploid human embryonic lung culture LECh-3 cells in a 96-well plate, characterized by preparing a two-percent agarose hydrogel in Dulbecco's buffer solution, forming microwells with a diameter of 500 μm in the wells of a 96-well plate, adding LECh-3 cell suspensions at a density of 20 × 10³ cells / ml to the formed microwells, culturing them at a temperature of +37°C in a humidified atmosphere with 5% CO2 for 48 hours until spheroids of LECh-3 cells are formed, followed by changing the DMEM nutrient medium with 10% FBS every 48 hours to maintain the viability of the spheroids.

[0022] The invention is explained with illustrations.

[0023] Fig. 1. Morphological analysis on the 4th day of the experiment: (a) - spheroids infected with HSV-1 / L2; (b) - spheroids without the virus (control). Images were obtained on a Zeiss Axio Vert.A1 inverted microscope (Carl Zeiss, Germany) with a Primo Plan-ACHROMAT 10× / 0.25 Ph1 objective and a WF 10× / 20 eyepiece.

[0024] Fig. 2. Evaluation of the result of culturing HSV-1 / L2 in spheroids by transferring the culture fluid onto the monolayer. (a) - CPE of the culture fluid collected from spheroids cultured with HSV-1 / L2 spheroids; (b) - absence of CPE of the culture fluid collected from spheroids without the virus (control). Images were obtained on a Zeiss Axio Vert.A1 inverted microscope (Carl Zeiss, Germany) with a Primo Plan-ACHROMAT 10× / 0.25 Ph1 objective and a WF 10× / 20 eyepiece.

[0025] Fig. 3. Comparative analysis of the adhesive properties of LECh-3 spheroids on a plastic surface 24 hours after transfer: (a) - control spheroid demonstrating attachment to the surface with the formation of flattened cells along the perimeter; (b) - HSV-1 / L2 infected spheroid that does not exhibit adhesive properties due to viral infection. Images were obtained on a Zeiss Axio Vert.A1 inverted microscope (Carl Zeiss, Germany) with a Primo Plan-ACHROMAT 10× / 0.25 Ph1 objective and a WF 10× / 20 eyepiece.

[0026] Implementation of the invention

[0027] LECH-3 cells were cultivated in DMEM medium containing 10% FBS in 25 cm² plastic flasks. A continuous monolayer formed on days 4-5 of cultivation at 37°C in a humidified atmosphere with 5% CO2. To disperse the cell layer during subcultures, a mixture of 0.25% trypsin and 0.02% Versene in a 1:3 ratio was used at 37°C. To increase the cell mass, three consecutive passages were performed at a 1:1 ratio.

[0028] To form microwells in the wells of a standard 96-well plate, a custom-designed mold was 3D-printed on a Form 3B+ printer using HarzLabs biocompatible photopolymer resin. The mold contained protrusions 500 µm in diameter and 2 mm in height, positioned according to the well geometry of the 96-well plate.

[0029] A 2% agarose hydrogel was prepared in Dulbecco's buffer. Agarose (HiMedia) was added to Dulbecco's buffer to a final concentration of 2% and heated in a water bath until the agarose was completely dissolved. The resulting hot 2% agarose hydrogel solution was added to the wells of a 96-well plate at a rate of 165 µl per well. A 3D-printed mold was then inserted into the plate so that the protrusions were positioned within the wells containing the agarose hydrogel. The plate was left at room temperature (22°C) for 5 minutes until the agarose hydrogel had completely solidified, after which the mold was carefully removed. The resulting 96-well plate with microwells in agarose hydrogel was subjected to UV irradiation with a wavelength of 254 nm and an intensity of 100 mW / cm² for 60 minutes for sterilization.

[0030] A suspension of LEC-3 cells was prepared in DMEM medium with 10% FBS at a density of 20 × 10³ cells / mL. The resulting suspension was added to the wells of a 96-well plate containing microwells in a 2% agarose hydrogel. The plates were incubated at 37°C in a humidified atmosphere with 5% CO2. After culturing LEC-3 cells in the agarose hydrogel microwells for 48 hours, single three-dimensional spheroids of LEC-3 cells formed in each well.

[0031] Further spheroid cultivation was carried out under the same conditions (temperature +37°C, humidified atmosphere with 5% CO2), with subsequent replacement of the DMEM culture medium with 10% FBS every 48 hours. Under this cultivation regime, LEC-3 cell spheroids maintained viability and structural integrity for at least 14 days, enabling their long-term use in experimental studies.

[0032] To evaluate the shape of spheroids, the roundness parameter was used, calculated using the formula:

[0033]

[0034] The morphometric parameters of the spheroids were calculated using the Spheroanalysis program. The analysis showed that the resulting LECh-3 cell spheroids are highly uniform in size and shape, confirming the technical result achieved—the formation of single LECh-3 cell spheroids of uniform size and shape in the wells of a standard 96-well plate and the high standardization of their morphometric characteristics.

[0035] To demonstrate the increased sensitivity of three-dimensional cell culture to biological agents, in particular to the herpes simplex virus type 1, the formed spheroids of LEC-3 cells were infected with the herpes simplex virus type 1 strain HSV-1 / L2 from the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology with an infectious activity of 6 lg TCID 50Further cultivation and evaluation of the cytopathic effect, infectious activity of the virus, and the viability of infected spheroids were carried out according to the methods given in the examples, which confirmed an increase in the sensitivity of the three-dimensional cell culture to the herpes simplex virus type 1.

[0036] Example 1: Preparation and culture of single spheroids from human diploid cells

[0037] LECH-3 cells were cultured in DMEM supplemented with 10% FBS in 25 cm² plastic flasks. A continuous monolayer formed on days 4-5 of culture at 37°C in a humidified atmosphere with 5% CO2. To disperse the cell layer during subcultures, a mixture of 0.25% trypsin and 0.02% Versene in a 1:3 ratio was used at 37°C. To increase the cell mass, three passages were performed at a 1:1 ratio.

[0038] To form microwells in a standard 96-well plate, a 2% agarose hydrogel was prepared in Dulbecco's buffer. Agarose (HiMedia) was added to Dulbecco's buffer to a final concentration of 2% and heated in a water bath until completely dissolved. The hot 2% agarose hydrogel solution was added to the wells of a 96-well plate at a rate of 165 µl per well.

[0039] Then, a specially designed 3D-printed mold containing 500 μm diameter, 2 mm high protrusions was inserted into the plate. The protrusions were positioned according to the geometry of the wells of the 96-well plate, such that the protrusions were located within the wells filled with agarose hydrogel. The plate was kept at room temperature (22°C) for 5 minutes until the agarose hydrogel completely solidified, after which the mold was carefully removed. The resulting 96-well plate with microwells in agarose hydrogel was then exposed to UV irradiation at a wavelength of 254 nm and an intensity of 100 mW / cm² for 60 minutes for sterilization.

[0040] A suspension of LEC-3 cells was prepared in DMEM medium with 10% FBS at a density of 20 × 10³ cells / mL. The resulting suspension was added to the wells of a 96-well plate containing microwells in a 2% agarose hydrogel. The plate was incubated at 37°C in a humidified atmosphere with 5% CO2 for 48 hours. After culturing LEC-3 cells in microwells of 2% agarose hydrogel for 48 hours, single three-dimensional spheroids of LEC-3 cells formed in each well.

[0041] Further spheroid cultivation was carried out at 37°C in a humidified atmosphere with 5% CO2, with subsequent replacement of the DMEM culture medium with 10% FBS every 48 hours. Under this cultivation regime, LEC-3 cell spheroids maintained structural integrity and viability for at least 14 days.

[0042] To evaluate the shape of spheroids, the roundness parameter was used, calculated using the formula:

[0043]

[0044] The calculation of morphometric parameters was carried out using the “Spheroanalysis” program.

[0045] After two days of cultivation, the diameters and roundnesses of 96 spheroids were plotted. The results demonstrated stable spheroid sizes with a coefficient of variation (CV) of 1.18% and a low interquartile range of 1.73% of the median. Analysis of spheroid shape revealed a median roundness value of 0.94 (where 1.0 is a perfect circle) with a CV of 1.68%. All roundness values ​​were within a narrow range from 0.927 to 0.961, and the interquartile range was 2.72% of the median. For both parameters (size and shape), a complete absence of statistical outliers, a high degree of data homogeneity, and stable reproducibility of the results were recorded.

[0046] LECH-3 cell spheroids remained stable for 14 days, with the DMEM culture medium containing 10% FBS being changed every 48 hours.

[0047] Thus, Example 1 demonstrates that when performing a sequence of actions corresponding to the claimed formula of the invention, the formation of single spheroids of LEC-3 cells, homogeneous in size and shape, in the wells of a standard 96-well plate is ensured and a high standardization of their morphometric parameters is achieved, which corresponds to the declared technical result.

[0048] Example 2: Cultivation of herpes simplex virus in spheroids of the LEC-3 cell line

[0049] LEC-3 cell spheroids were formed using the method described in Example 1, implementing the sequence of steps specified in the claims. After the formation of stable, single, three-dimensional spheroids of LEC-3 cells, they were infected with herpes simplex virus type 1 (HSV-1) for 48 hours.

[0050] The following steps were performed for infection. The growth medium was removed from the wells containing the cell spheroids. A viral suspension of the herpes simplex virus type 1 strain HSV-1 / L2 was prepared from the State Virus Collection of the D.I. Ivanovsky Institute of Virology with an infectious activity of 6 lg TCID. 50 in maintenance medium (DMEM without serum). 100 µl of viral suspension were added to each well so that the final virus concentration in the well was 10² TCID 50 / ml.

[0051] Immediately after infection, the plates were placed in an incubator at 37°C and 5% CO2, where the HSV-3 cell spheroids were cultured for 5 days. They were observed daily under an inverted microscope to assess the cytopathic effect (CPE) caused by the virus. On the fourth day after infection, significant changes in the structure of the HSV-1-infected spheroids were observed: the spheroids became less compact and their integrity was compromised, indicating viral replication and progression of the viral infection. Control (uninfected) spheroids retained their structural integrity and density (Fig. 1).

[0052] After completion of the incubation period, virus replication was assessed by the following methods.

[0053] Titration of culture fluid:

[0054] The culture fluid (CF) was collected from wells with infected spheroids, after which serial dilutions were carried out from 1 / 2 to 10 -7and applied the resulting dilutions to a monolayer of LEC-3 cells in triplicate, followed by incubation for 5 days under standard culture conditions (+37°C, 5% CO2). When evaluating the results, a clear correlation was observed between the degree of cytopathic effect and the dilution of the virus-containing fluid: in dilutions of 1 / 2, 1 / 10, and 10 - ² Complete CPE was noted in all replicates (++++) (Fig. 2), at a dilution of 10 - ³ A slightly less pronounced CPE was observed in all three replicates (+++−), at a dilution of 10 -4 CPE was moderately expressed (++−−), in a dilution of 10 -5 individual foci of CPE were detected, affecting approximately 30% of the cell monolayer (+−−−), and in dilutions of 10 -6 and 10 -7 CPE foci were minimal, affecting no more than 20% of the monolayer (±−−−). In control samples (CF from wells with uninfected spheroids), CPE was not observed in any replicate.

[0055] The obtained data indicate the preservation of high replicative activity of the herpes simplex virus type 1 in three-dimensional spheroids of LEC-3 cells and confirm that the formed spheroids provide sufficient conditions for the effective replication of the virus and the subsequent detection of its infectious activity in a standard test system on a monolayer of LEC-3 cells.

[0056] Spheroid viability assessment:

[0057] To assess viability, 4 spheroids from each experimental (infected) and control (uninfected) group were transferred to the plastic surface of a culture flask in growth medium (DMEM with 10% FBS). After 24 hours, control spheroids attached to the surface, forming flattened cells around their perimeter. However, infected spheroids did not attach to the surface, indicating their nonviability due to viral infection (Fig. 3).

[0058] Thus, Example 2 demonstrates that LECH-3 cell spheroids obtained according to the claimed method exhibit high sensitivity to the biological agent herpes simplex virus type 1 (HSV-1 / L2), ensuring its effective replication and detection of infectious activity. This confirms the achievement of the claimed technical result in terms of increasing the sensitivity of the three-dimensional cell culture to biological agents.

[0059] Advantages of the proposed method:

[0060] Improved spheroid formation: The method enables the production of single spheroids in each well of a 96-well plate with high reproducibility in size and shape.

[0061] Physiological relevance: 3D spheroids provide more natural conditions for cellular interactions and functioning, which is especially important when modeling viral infections.

[0062] Availability and cost-effectiveness: the use of the domestic LEC-3 cell line and standard laboratory materials makes the method accessible to a wide range of research laboratories.

[0063] The problem is solved by the fact that the proposed method for culturing single spheroids of LEC-3 cells in a 96-well plate uses a specially designed form manufactured by 3D printing on a Form 3B+ printer using a biocompatible photopolymer resin HarzLabs, containing protrusions with a diameter of 500 μm and a height of 2 mm, located in accordance with the geometry of the wells of the 96-well plate, which ensures the formation of standardized microwells in a 2% agarose hydrogel prepared in Dulbecco's buffer solution. Adding a suspension of LECH-3 cells with an optimized density of 20 × 10³ cells / ml to the resulting microwells and subsequent cultivation at a temperature of +37°C in a humidified atmosphere with 5% CO2 for 48 hours makes it possible to obtain single three-dimensional cell aggregates of a high degree of homogeneity in each well.Regular replacement of the DMEM culture medium with 10% FBS every 48 hours ensures long-term viability of spheroids for 14 days, which makes it possible to conduct a variety of experimental studies, including high-throughput screening using standard laboratory equipment with significant savings in cell material and reagents.

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