CN117903940A - Culture apparatus, method for processing incubator, and cell culture method - Google Patents
Culture apparatus, method for processing incubator, and cell culture method Download PDFInfo
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- CN117903940A CN117903940A CN202410090207.6A CN202410090207A CN117903940A CN 117903940 A CN117903940 A CN 117903940A CN 202410090207 A CN202410090207 A CN 202410090207A CN 117903940 A CN117903940 A CN 117903940A
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Abstract
The invention discloses a culture device, a processing method of a culture device and a cell culture method. The culture device includes a culture cassette having a receiving space and an open top, and a culture vessel. The incubator is made of polydimethylsiloxane and is located in the accommodating space, the bottom surface of the incubator is tightly attached to the bottom wall of the incubator, the side wall of the incubator is tightly attached to the inner side wall of the incubator, the incubator is provided with an incubation groove, an input hole and an output hole, and the incubation groove is formed by recessing of the bottom surface of the incubator. The input hole is formed by a top wall of the culture tank being recessed to a top surface of the incubator. The output hole is formed from the top wall of the culture tank to the top surface of the incubator in a recessed mode and is arranged at intervals with the input hole.
Description
Technical Field
The invention relates to the technical field of cell culture devices, in particular to a culture device, a processing method of a culture device and a cell culture method.
Background
With the rapid development of tissue engineering and regenerative medicine, more and more researches require isolated culture and expansion of primary cells. However, the conventional primary tissue isolation and stem cell culture methods have various problems such as low cell viability, unstable fixed tissue methods, and low clone formation efficiency, and if the primary culture of a minute amount of tissue is performed, the success rate is lower. Conventional methods require large amounts of media and reagents, increase the cost of the experiment, and limit the feasibility of large-scale experiments. Therefore, researchers need to seek more efficient and cost-effective culture methods to improve experimental efficiency and reduce resource consumption.
Disclosure of Invention
In order to solve the above technical problems, embodiments of the present invention provide a culture apparatus, a processing method of a culture vessel, and a cell culture method to solve the above problems.
To solve the above technical problems, embodiments of the present invention provide a culture apparatus, including:
a culture cassette having an accommodation space and an opening at a top; and
The incubator, the incubator is made by polydimethylsiloxane and is located in accommodation space, the bottom surface of incubator is hugged closely the diapire of cultivateing the box, the lateral wall of incubator is hugged closely the inside wall of cultivateing the box, the incubator is equipped with:
a culture tank formed by recessing a bottom surface of the incubator;
An input hole formed by a top wall of the culture tank being recessed to a top surface of the incubator; and
And the output hole is formed from the top wall of the culture tank to the top surface of the incubator in a recessed mode and is arranged at intervals with the input hole.
The invention also relates to a processing method of the incubator, which comprises the following steps:
s1, mixing a silicon polymer and a cross-linking agent in proportion, and uniformly stirring to form a mixed colloid;
S2, extracting bubbles in the mixed glue;
S3, superposing a sheet-shaped mold on the bottom wall of the culture box, pouring the mixed colloid after air bubble extraction into the culture box, and heating and drying until the mixed colloid is solidified and molded to form a incubator;
S4, after the incubator is taken out, separating the die from the incubator, and forming a culture tank on the bottom surface of the incubator; and punching an input hole and an output hole on the top wall of the culture tank by using a puncher, and enabling the input hole and the output hole to penetrate through the culture tank respectively.
In one embodiment, the culture cassette is a petri dish and the mold is a slide.
In one embodiment, in step S2, a plurality of the molds are respectively stacked on the bottom wall of the culture box and are arranged at intervals;
In step S4, a plurality of culture tanks are formed by separating the plurality of molds from the incubator, and the input holes and the output holes are punched in the top walls of the culture tanks by a puncher.
In one embodiment, at least two of the molds are stacked in a vertical direction.
In one embodiment, at least two of the molds are different in size.
In one embodiment, step S3, heating is performed at a temperature of 70 ° -80℃for a period of 60min-70min.
The invention also relates to a cell culture method, comprising the following steps:
a. cleaning the culture device of claim 2 to remove impurities and residues on the surface of the culture device;
b. sterilizing the incubator and the culture box; c. placing the incubator within the incubator;
d. The culture medium is moved from the inlet port into the culture tank.
The cell culture method of claim 8, wherein the incubator is made of polydimethylsiloxane; in the step a, the incubator is placed in phosphate buffer salt solution for soaking for a preset time, impurities and residues on the surface of the incubator are removed, and then the incubator is placed in deionized water for ultrasonic cleaning, so that the cleanliness of the surface of the incubator is ensured.
In one embodiment, in step b, after sterilizing the incubator, the incubator is also wiped dry using a sterilizing filter paper.
The present invention addresses these problems by developing an incubator made of elastic transparent material for fixing tissues and culturing primary cells. The device adopts the culture tank of small volume and the material of gas permeability, can reduce the pollution risk to reduce the consumption of culture medium. The device can be used for better fixing micro tissues, can improve the survival rate and proliferation rate of primary cells, and simultaneously provides micro environment conditions for the tissues, which are more similar to the primary state. By optimizing the fixing and culturing method, the invention is expected to improve the tissue fixing and culturing effect and provides a more reliable experimental platform for cytology and biology research. In addition, the small-sized culture tank can reduce the use of culture medium and containers, thereby reducing the cost.
Drawings
FIG. 1 is a schematic diagram of an incubator according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view of the incubator of FIG. 1.
FIG. 3 is a schematic view of an incubator according to another embodiment of the present invention.
FIG. 3a is a schematic view of an incubator according to another embodiment of the present invention.
FIG. 4a is the medium composition of a primary culture of mouse brain tissue.
FIG. 4b is a graph of cell growth on day four of primary culture of mouse brain tissue in three gauge incubators and 24 well plates.
FIG. 5 is a graph comparing the amounts of harvested cells from primary cultures of mouse brain tissue in three gauge incubators and 24 well plates.
FIG. 6a is a graph of cell harvest number versus total cell number in three gauge incubators and 24 well plates per unit volume.
FIG. 6b is a graph of cell harvest versus total cell count for three gauge incubators and 24 well plates per unit volume of medium.
FIG. 7 is a graph showing the effects of neurosphere culture on primary cultured cells of brain tissue of mice on day eight in three gauge incubators and 24 well plates.
FIG. 8 is a culture of dental pulp tissue in a 24-well plate and a 0.17mm deep culture tank.
FIG. 9 shows the positive cloning efficiency and cell number per unit volume and unit area of dental pulp tissue cultured in 24-well plates and 0.17mm deep culture tanks.
FIG. 10 is a culture of dental pulp tissue in 96-well plates and 0.2mm deep culture tanks.
FIG. 11 shows the culture conditions and the cell climbing rate of dental pulp tissue in a 96-well plate and a 0.2mm deep culture tank.
FIG. 12 is the cell count of dental pulp tissue in 96-well plates and 0.2mm deep culture tanks.
FIG. 13 is a graph showing the cell climbing rate of dental pulp tissue in a 96-well plate and a 0.2mm deep culture tank.
FIG. 14 is the cell count of dental pulp tissue in 96-well plates and 0.2mm deep culture tanks.
FIG. 15 is cells in 96-well plates and 0.2mm deep culture tanks on day six of dental pulp tissue.
Reference numerals: 100. a culture vessel; 1. a culture tank; 2. an input hole; 3. and an output hole.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application will be described in detail below with reference to the accompanying drawings. However, those of ordinary skill in the art will understand that in various embodiments of the present application, numerous technical details have been set forth in order to provide a better understanding of the present application. The technical solutions claimed in the claims of the present application can be realized without these technical details and various changes and modifications based on the following embodiments.
Throughout the specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to be open-ended, meaning of inclusion, i.e. to be interpreted to mean "including, but not limited to.
The following detailed description of various embodiments of the present invention will be provided in connection with the accompanying drawings to provide a clearer understanding of the objects, features and advantages of the present invention. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the invention, but rather are merely illustrative of the true spirit of the invention.
Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise.
In the following description, for the purposes of clarity of presentation of the structure and manner of operation of the present invention, the description will be made with the aid of directional terms, but such terms as "forward," "rearward," "left," "right," "outward," "inner," "outward," "inward," "upper," "lower," etc. are to be construed as convenience, and are not to be limiting.
The present invention relates to a culture apparatus including a culture cassette having a receiving space and having an open top and a culture vessel 100 thereof, the culture cassette optionally including but not limited to a 24-well plate, a 96-well plate, a 12-well plate, a 6-well plate, a culture dish, etc., and the culture apparatus 100, and the present invention is not limited to the specific embodiment of the culture cassette.
The incubator 100 is made of elastic transparent materials, wherein the elastic transparent materials comprise rubber, preferably polydimethylsiloxane, and the polydimethylsiloxane is nontoxic and has the characteristics of ultrahigh molecular weight, low viscosity, unique fluidity and the like, and the incubator 100 made of the polydimethylsiloxane also has certain toughness, good compatibility and high transparency, and is very suitable for culturing cells.
The incubator 100 can be placed in a petri dish or six incubators 100 can be placed in six wells of a 6-well plate, respectively. Taking a culture dish as an example, the bottom surface of the incubator 100 is closely attached to the bottom wall of the culture dish, and the side wall of the incubator 100 is closely attached to the inner side wall of the culture cassette.
Specifically, the incubator 100 may be a column, such as a cylindrical structure or a quadrangular prism structure, and the present invention is not limited to the specific structure of the incubator 100, and the structure of the incubator 100 needs to be matched with the accommodating space of the culture cassette.
In the embodiment shown in FIGS. 1 and 2, the incubator 100 is a cylindrical structure that can be placed in a culture dish. The incubator 100 may be sized according to the size of the outer diameter of the dish, i.e., the outer diameter of the incubator 100 needs to match the inner diameter of the dish. The height of the incubator 100 is preferably less than the depth of the dish, and the height of the incubator 100 is preferably in the range of 0.5cm to 1cm, including but not limited to 0.5mm, 0.6cm, 0.7cm, 0.8cm, 0.9cm, and 1cm. The proper thickness facilitates demolding of the incubator 100 from the culture dish during manufacture of the incubator 100, and allows for a tight, threaded placement of the incubator 100 into the culture dish with substantially no medium communication between each culture tank. The incubator 100 is provided with a culture tank 1, an input hole 2 and an output hole 3, wherein the culture tank 1 is formed by recessing the bottom surface of the incubator 100, the inner wall of the culture tank 1 can be in a quadrangular prism or other cylinder structure, the depth of the culture tank 1 can be set according to the needs, and the invention is not limited to the specific shape of the culture tank 1.
In the embodiment of FIGS. 1and 2, the culture tank 1 has a cylindrical structure. The depth of the culture tank 1 is preferably in the range of 0.1mm to 2mm, optionally including 0.1mm、0.2mm、0.3mm、0.4mm、0.5mm、0.6mm、0.7mm、0.8mm、0.9mm、1mm、1.1mm、1.2mm、1.3mm、1.4mm、1.5mm、1.6mm、1.7mm、1.8mm、1.9mm and 2mm. In addition, in the embodiment shown in FIGS. 1and 3, three culture tanks 1 are provided on the bottom surface of the incubator 100, and the three culture tanks 1 are provided at uniform intervals and have the same shape. It should be understood that more culture tanks 1 may be provided, and the shapes of the culture tanks 1 may be different, and the depths of the culture tanks 1 may be set as required to adapt to different cell culture requirements.
As a preferred embodiment, as shown in FIG. 3, each of the three culture tanks 1 has an elongated quadrangular prism structure, and the length of each culture tank 1 is longer than the height thereof. The length of the culture tank 1 may be set according to the radial dimension of the incubator 100 as long as it is not greater than the radial dimension of the incubator 100. The width of the culture tanks 1 may be set according to the number of the culture tanks 1, and the plurality of culture tanks 1 may be provided at intervals so as not to exceed the outer diameter of the incubator 100. In the embodiment shown in FIG. 3a, a part of the culture tanks 1 has a rectangular prism structure, and the other part has a cylindrical structure in the embodiment shown in FIG. 1. Of course, in other embodiments, the shape of the culture tank 1 may be set to other shapes, and the present invention is not limited to the specific shape of the culture tank 1.
The input hole 2 is a through hole extending in the vertical direction, and the input hole 2 may be regarded as a through hole formed by the top wall of the culture tank 1 being recessed to the top surface of the incubator 100. The input hole 2 is a passage of the culture medium into the culture tank 1, and the culture medium can be fed from the input hole 2 into the culture tank 1 by a pipette. The diameter range of the input hole 2 is preferably 0.5mm-1.5m, in the pipetting process, the pipette can prop up the input hole 2, after pipetting, the input hole 2 can be reduced after being extruded, so that culture medium can be conveniently moved into the culture tank 1 easily, and excessive air or impurities can be prevented from entering the culture tank 1 from the input hole 2.
The outlet port 3 and the inlet port 2 are formed in a substantially uniform manner from the top wall of the culture vessel 1 to the top surface of the incubator 100. The output hole 3 is arranged at intervals with the input hole 2 and has a diameter larger than that of the input hole 2. In the cell culture process, the nutrient solution needs to be replaced, and when the nutrient solution is replaced, the nutrient solution can be added into the culture tank 1 from the input hole 2 through the pipetting gun, and the old nutrient solution is discharged from the output hole 3. So that the diameter of the outlet opening 3 is larger than the diameter of the inlet opening 2 in order to facilitate smooth discharge of the liquid from the outlet opening 3. As a preferable scheme, the diameter of the output hole 3 ranges from 1.5mm to 2.5mm, the output hole 3 in the range can smoothly discharge liquid, excessive evaporation of culture medium can be avoided in the cell culture process, and impurities can be prevented from entering the culture tank 1 from the output hole 3.
Each culture tank 1 is communicated with one input hole 2 and one output hole 3, each input hole 2 and output hole 3 are often formed by recessing the top wall of the corresponding culture tank 1 to the top surface of the incubator 100, and the input hole 2 of each culture tank 1 is arranged at intervals from the output hole 3. That is, the number of input holes 2, output holes 3 and culture tanks 1 corresponds one by one.
The invention also relates to a processing method of the incubator 100, which specifically comprises the following steps:
S1, mixing a silicon polymer and a cross-linking agent in proportion, and uniformly stirring to form a mixed colloid, wherein the proportion of the silicon polymer to the cross-linking agent is generally 10:1;
S2, vacuum pumping is carried out on the mixed colloid to remove bubbles in the mixed colloid, so that the mixed colloid is ensured to be free of bubbles;
S3, superposing the flaky mold on the bottom wall of the culture box, and injecting the mixed colloid after air bubble extraction into the culture box, namely, covering the flaky mold by the mixed colloid, and heating the flaky mold in a metal heat block at the temperature of 70-80 ℃ for 60-70 min. Heating and drying until the mixed colloid is solidified and molded to form the incubator 100;
The thickness of the mixed gel injected into the culture cassette mainly affects the thickness of the incubator 100, that is, the height of the incubator 100, and the thickness of the incubator 100 determines the hardness thereof, so that the smaller the thickness of the incubator 100 is, the softer the incubator is, and the subsequent operations such as demolding and punching are easier. The larger the thickness of the incubator 100, the harder it is, and the inconvenience of demolding is, but each well can be better divided, ensuring independence of each well. The thickness of the incubator 100 is preferably 0.5cm to 1cm.
S4, after taking out the incubator 100, separating the mold from the incubator 100, separating the cured incubator 100 from the culture cassette, carefully removing the incubator 100 without damaging the culture cassette, and carefully taking out the mold using tweezers. After the mold is taken out, the bottom surface of the incubator 100 forms a culture tank 1; an input hole 2 and an output hole 3 are punched to the top wall of the culture tank 1 with a puncher, and the input hole 2 and the output hole 3 are made to penetrate the incubator 100, respectively.
In step S4, a plurality of molds are separated from the incubator 100 to form a plurality of culture tanks 1, and the top walls of the culture tanks 1 are perforated with a puncher to form the input holes 2 and the output holes 3, respectively.
In step S3, the culture cassette may be a multi-well plate or a culture dish. The selection of appropriate outer mold sizes ensures compatibility of the mold with the cell culture apparatus and provides adequate culture area.
Taking the culture dish as an example, the sheet-shaped mold may be selected from glass slides or other metal sheets, and a plurality of glass slides may be placed on the bottom wall of the culture dish at intervals, and two, three or more glass slides may be stacked in the vertical direction in order to increase the thickness of the culture tank 1. Of course, it is also possible to stack some of the slides and other individual slides on the bottom wall of the culture dish to form culture tanks 1 of different depths. Different sizes of molds may be used to form different sizes of culture tanks 1. The size and shape of the mould can be adjusted to customize the mould according to the size and shape of the tissue so as to adapt to different cell culture requirements.
The thickness of the incubator 100 can be adjusted by controlling the amount of the mixed colloid. Different thicknesses may have an effect on the performance and degree of deformation of the incubator 100. Thicker incubators 100 are simpler to operate and reduce evaporation of the medium during incubation, while also being less prone to deformation problems. Accordingly, in manufacturing the incubator 100, an appropriate thickness of the incubator 100 can be selected according to experimental requirements and convenience of operation. By adjusting parameters of the incubator 100 according to the requirements of different tissues, the incubator 100 can be customized for a particular experiment. These variable parameters can be flexibly adjusted according to experimental requirements to ensure that the performance and use of the incubator 100 meet the goals and requirements of the study.
The invention also relates to a cell culture method, comprising the following steps:
a. Using the above-mentioned configuration device, firstly cleaning the incubator 100 and the incubator box to remove impurities and residues on the surface of the incubator 100; for the incubator 100 made of polydimethylsiloxane, the incubator 100 may be cleaned with a phosphate buffer solution, the incubator 100 may be immersed in the phosphate buffer solution for a preset time to remove impurities and residues on the surface of the incubator 100, and then the incubator 100 is placed in deionized water for ultrasonic cleaning to ensure the cleanliness of the surface of the dish.
B. The incubator 100 and the incubator box are subjected to high-temperature sterilization treatment, so that microorganisms remained on the surface of the mold can be effectively killed, and the risk of pollution is reduced; taking out the incubator 100 and the incubator box after high-temperature sterilization, and lightly wiping the incubator box with sterile filter paper to ensure the surface to be dry, wherein the operation can reduce bubbles generated in a culture tank in the subsequent liquid adding process;
cells with special culture surface requirements can be coated with cell adhesive agent on the inner wall of the culture box, wherein the cell adhesive agent can be collagen or other cell adhesive substances so as to enhance the attachment and growth of the cells;
c. placing the incubator 100 in the incubator, which may be a dish or an orifice plate, with sterile forceps, ensuring that the incubator 100 is properly attached to the orifice plate or the inner wall of the dish and that there is no gap or looseness;
d. The culture medium is moved from the inlet port into the culture tank 1. Specifically, the tip of the pipette is accurately inserted into the bottom of the input port with the tip of the pipette ensured to be positioned inside the culture tank 1. Culture medium of a previously adjusted density was fed from the inlet port 2, taking care to control the volume fed to ensure that the culture medium fills the entire culture tank 1.
During the addition of the medium, care is taken to empty the air bubbles to ensure that the medium fills the culture tank 1 and is in good contact with the cells. The removal of bubbles may be aided by slow infusion and gentle shaking of incubator 100. Ensure that all the culture tanks 1 are filled with medium and that a uniform distribution of medium is ensured.
By strictly following the aseptic technique, proper installation of incubator 100 and addition of tissue-containing medium ensures that the cells are well-conditioned in culture tank 1. The implementation of these steps will help reduce contamination and cell damage while improving the reliability and reproducibility of the experiment.
The anchorage capability of primary tissue is often one of the key factors in successful cell culture and expansion during cell culture. Many primary tissues, especially tissues derived from adult animals or humans, have the problem of poor adhesion. Conventional adherent culture methods are not effective in isolating and expanding stem cells in tissue, and conventional primary cell anchoring methods (e.g., glass sheet pressing) may result in tissue damage and cell inactivation. In addition, conventional methods of fixing tissues and primary cells may cause damage to tissue structure and cell integrity, affecting the accuracy of the experimental results. To improve the reproducibility and reliability of the experiment, we developed an incubator 100 to reduce tissue and cell damage and develop cell culture methods that enhance adherence to better preserve the original properties of the tissue and increase cell viability and proliferation capacity.
For primary culture of stem cells, many factors such as extracellular matrix (ECM) components, cell-cell interactions, oxygen, nutrients, etc. need to be considered to ensure that the stem cells retain their characteristics and function. The incubator 100 of the present invention can be provided with a plurality of small-sized incubators 1, and the small-sized incubators 1 can provide a culture environment of smaller volume and more similar to that of the original state, which helps to more accurately simulate in vivo conditions. According to Bersini et al, the small volume culture device can better simulate factors such as extracellular matrix components, cell-cell interactions, oxygen levels, nutrient supplies and the like, and promote more realistic experimental results and research findings. Meanwhile, for some primary tissues, especially human tissues with small tissue quantity, the traditional large-volume adherent culture still has more limitations in the aspects of tissue adherence, culture medium volume and the like.
Conventional tissue fixation and conventional volume culture have a number of disadvantages. For example, slide adherence culture requires pressing the tissue sample under the slide, which can easily lead to mechanical trauma and death of the cells, while hindering nutrient and gas exchange. In addition, this method requires a large amount of culture medium and a limited culture area, increasing costs and operational trouble. In conventional volumetric culture, the relative tissue mass of the culture medium is large, resulting in a sparse arrangement of tissue cells, affecting interactions and communication between cells, especially for proliferation and maintenance of stem cells.
In view of these problems, the present invention has developed an incubator 100 made of elastic transparent material for fixing tissues and culturing primary cells. The device adopts the culture tank 1 with small volume and the material with air permeability, can reduce pollution risk to reduce the consumption of culture medium. The device can reduce the mechanical trauma and death rate of cells, provide micro-environment conditions which are more similar to the original state, improve the survival rate and proliferation rate of primary cells, and provide micro-environment conditions which are more similar to the original state for tissues. By optimizing the fixing and culturing method, the invention is expected to improve the tissue fixing and culturing effect and provides a more reliable experimental platform for cytology and biology research. In addition, the small-sized culture tank 1 can reduce the use of a culture medium and a container, thereby reducing the cost.
The following two experiments were designed to verify the effect of cell culture in the incubator 100 of the present invention.
Experiment one: the brain tissue of the mice was cultured.
In the culture of primary tissue, the tissue amount is very important to the success rate of primary culture. First, culture tests were performed with a large number of brain tissues easily obtained, and the effect of the incubator 100 on the culture of primary cells was observed when the success rate was high. We used mouse hippocampal tissue within 24 hours of birth to isolate neural progenitor cells (NPC for short), which are abundant in hippocampal tissue and were primary cultured to high power.
The following processes of mouse material taking and tissue pretreatment and culture are as follows:
1. preparation of mouse NPC medium with composition as shown in fig. 4a, P0 neonatal mouse was prepared: pregnant mice with codes of C57BL/6J are selected, and the sea horse tissues of the newborn mice are obtained within 24 hours after the pregnant mice are produced.
2. Newborn healthy mice were sterilized with 75% (volume fraction) alcohol, sacrificed by cervical removal under aseptic conditions, scalp and skull were cut off, brain tissue was removed, and placed in plates (ice box under) containing 1% penicillin-streptomycin mixed solution in Du's phosphate buffer (DPBS buffer for short).
3. Hippocampus tissue was isolated aseptically. The brain tissue was kept with its back facing upwards, the cerebral cortex was carefully turned open under the mirror, the hippocampus was exposed, the tissue around the hippocampus was separated with an ophthalmic scissors or a pointed forceps, and the tissue was taken out and placed in a plate containing 2# medium.
4. The tissue is sheared into tissue blocks smaller than 1mm 3 by iris scissors, ground and filtered by a sterile 200-mesh screen, the volume of the obtained tissue is quantified, and the tissue is resuspended to a working concentration by using a No. 2 culture medium after the quantification.
5. The prepared sterile incubator 100 is placed in a incubator, which employs a 6-well plate to ensure that no liquid is present in the incubator 100's incubation well and that the lower edge of the incubator 100 is completely flush with the bottom wall of the well. Appropriate amounts of tissue were added to the culture tank 1, and a small amount of tissue culture groups diluted 2-fold and 5-fold in the incubator 100 and corresponding normal condition control groups in the 24-well plate were set, respectively, for comparison test. Further, in this experiment, three specifications of incubators 100 and 24-well plates were used as a comparative experiment, and the depths of the culture tanks of the three specifications of incubators 100 were 0.2mm, 0.5mm and 1mm, respectively.
The liquid is changed every three days in the culture process, so that the nutrition required by the growth of the cells is ensured.
Subculture and identification of NPC
FIG. 4b shows the cell growth conditions of the 0.2mm, 0.5mm and 1mm culture tanks and 24 well plates after observing the depth of the cell growth on the fourth day of the primary culture of the brain tissue of the mice, and when the inoculation amount of the brain tissue is 2.5uL, a large amount of cells grow out in the culture devices 100 and 24 well plates of three specifications, and the concentration is the highest tissue culture concentration, as shown in FIG. 4 b. When the inoculation amount of brain tissue was 0.5uL, it was found that the cells in the 24-well plate grew unevenly and had many voids, while the cells in the culture tank 1 of the incubator 100 grew evenly, and the cell confluency reached 80% or more. When the inoculation amount of brain tissue is 0.25uL, the growth state of cells in a 24-well plate is poor, and the cells are scattered and distributed mostly, and the cells are rarely climbed out around a tissue block. Cells grow around more than tissues in the culture tank, the confluence is higher than that of a 24-pore plate, the morphology is uniform, and the distribution is uniform.
FIG. 5 is a summary comparison of the number of P0 cells in each group of three replicates, and it can be seen that the 2.5ul tissue mass group, the most cells obtained in the 24 well plate; in the case of a sharp decrease in tissue mass, the 0.25ul tissue mass group, however, harvested more cells than the 24 well plate group in each incubator 100 group.
Fig. 6a and 6b are comparison of the cell harvest per unit area and unit volume of the primary culture of murine brain tissue, and further analysis of the data shows that the cell harvest number is similar to the trend of the total cell harvest number in the unit body area, as shown in fig. 6a. Whereas the low volume culture cell protocol had a higher cell mass per unit volume of medium, the 0.25ul tissue mass groups were more than 30-fold different as shown in FIG. 6b.
Following is a spheronization of mouse NPCs to verify cell purity.
And carrying out passage adherence culture on the primary cultured cells, and purifying the cells three times in succession to obtain single cells. The harvested cells were cultured in 24-well plates subjected to tissue culture treatment (abbreviated as TC treatment), and FIG. 7 shows the culture of neurospheres on the eighth day, and it was found from the results that both the cells cultured in the culture apparatus 100 and the 24-well plates were subjected to suspension culture to form neurospheres, which demonstrated that the purity of NPC was within an acceptable standard range, and that the culture apparatus 100 did not significantly affect the differentiation ability and characteristics of the cells.
In this section of the experiment, we compared cells cultured using the incubator 100 with a conventional 24-well plate, and found that the incubator 100 exhibited a significant advantage at low tissue amounts, enabling more cells to be obtained. In addition, the use of the incubator 100 promotes uniform growth of cells, increases cell confluence, and makes cell distribution more uniform. This is important to maintain cell status, reduce cell loss, and ensure consistency of the experiment. Another significant finding is that at the same volume, the incubator 100 obtains a greater number of cells, which means that under the same experimental conditions, the incubator 100 requires less medium, thereby saving experimental costs. Furthermore, we speculate that the use of incubator 100 may create a better cellular microenvironment, promoting cell proliferation.
Experiment II: dental pulp stem cell culture
The dental pulp mesenchymal stem cell is a kind of pluripotent stem cell existing in the dental pulp tissue of the human body, has a high regeneration capacity and a multi-directional differentiation potential, and thus is widely used in the fields of tissue engineering, regenerative medicine, and the like, and thus the present invention also uses the dental pulp tissue to verify the effect of the incubator 100.
The device before culture:
Preparation of incubator 100: first, a size-adapted incubator 100 is customized according to the characteristics of the dental pulp tissue of the mouse, and then subjected to demolding and autoclaving. The high-pressure sterilization can effectively kill bacteria and viruses on the surface of the die, and reduce the pollution risk of cell culture.
Pretreatment of culture: the sterilized incubator 100 is stored in pure water or phosphate buffer (PBS for short), the incubator 100 is taken out in advance, and surface moisture is sucked dry with sterile filter paper, because water droplets are contained to introduce bubbles when the medium is subsequently added.
The following are mouse dental pulp materials, tissue pretreatment and culture processes:
Tissue washing and separation: the mice were sacrificed by cervical removal, the heads were cut and the lower incisors (including mandibles) were dissected from the mice, surrounding tissues were carefully removed, and contamination of other tissues was reduced.
The mouse incisors were placed in PBS containing antibiotics diluted to working concentration and washed multiple times to remove blood stains and contamination from the surface. The bone portion is carefully pried open at the root portion with a surgical knife along the shape of the tooth, exposing the pulp tissue therein. The distal pulp tissue was carefully removed, leaving a larger portion of the root stem cells, approximately 1mm-2mm.
Sufficient tissue was collected, transferred to a 1.5ml centrifuge tube lid, and about 10ul of complete medium (medium+20% fetal bovine serum+1% penicillin-streptomycin mix) was added dropwise, the tissue was chopped as much as possible with micro-scissors, then about 200ul of medium was added, collected in a centrifuge tube, and stored on 4 ° or ice for use.
Cell culture in incubator 100: the culture vessel 100 dried in advance was placed in a petri dish, and the separated dental pulp tissue suspension was added to the culture vessel 100 (the depth of the culture vessel was 0.17 mm) and 24 wells of the control at 30 ul/well, respectively, and the medium was carefully added to prevent the occurrence of air bubbles in the culture vessel.
During the culture, the medium was changed about every 3 days to provide enough nutrition to support proliferation and differentiation of cells, and the cells were observed and recorded. If the medium evaporates faster, it can also be placed in a wet box, which is then placed in an incubator.
Cells cultured in incubator 100 were compared with a conventional adherent culture control to evaluate the effect of incubation in incubator 100.
Cell digestion and passage: when the cells grow to a sufficient number, digestive passages are required. Upon digestion, the cells may be separated from the culture dish or incubator 100 using diluted trypsin or other digestive enzymes and then transplanted into a new dish for the next round of culture. During passage of cells, care is taken to control the cell density and digestion time to ensure healthy growth and stability of the cells.
And (5) subculturing and culturing dental pulp stem cells for identification.
Primary cultured multi-tissue and cell morphology: after the tissue suspension is added to the 0.17mm deep culture tank and 24-well plate of incubator 100, the tissue fragments settle to the bottom of the dish. Compared to 24-well plates, the incubator 100 better immobilizes tissue, thereby promoting the climbing of adherent stem cells out of the tissue. As shown in FIG. 8, at various time points of culture, cells in the incubator 100 crawl out faster and clone sizes are significantly larger. On the next day, it has been seen that tissue of the incubator 100 group begins to crawl out of cells, while on the fifth day more cells have proliferated.
On day 12 after culture, the total positive clone numbers were calculated, and the comparison found that the tissues in the culture tank all crawled out stem cell clones at about 40% of the tissues, while the positive clone rate of the 24-well plate was only about 8% -25%, as shown in fig. 9A. When the clones were proliferated until passaging was possible (typically 10-13 days), cells were digested and counted, and the culture tank could reach about 6×10 4/cm2 as shown in fig. 9B, whereas the total tissue yield in 24-well plates was 3×10 4/cm2, and the difference could reach 2-fold. According to the relative volumes, as shown in fig. 9C, the volume in the incubator 100 can reach 1.08×10 7/ml, whereas the volume in the 24-well plate culture is only 2.8×10 5/ml, which is different by about 50 times.
To further verify the effect of incubator 100 on primary tissue, a comparison of 96-well and 0.2mm thick culture tanks was added, each 96-well and 0.2mm thick culture tank was filled with a piece of rat dental pulp tissue, and the degree of climbing out of primary cells, the ratio, and the number of acquired cells that have been subsequently.
As clearly seen from the image in FIG. 10, the cells in the culture tank grew faster, more in number and more in cloned area than in the 96-well conventional adherent culture.
FIG. 11 shows the culture conditions of dental pulp tissue in a 96-well plate and a culture vessel 100 and the cell climbing rate. FIG. 12 is a graph showing cell numbers of dental pulp tissue in 96-well plates and incubators 100. FIGS. 13 and 14 show the cell climbing-out ratio and the cell harvest number of the well plate and the incubator 100, respectively. FIG. 15 shows the cell culture in 96-well plates and incubators 100.
On day 6 after culture, the total positive clone numbers were calculated, and the comparison shows that the rat tissues of the 100 groups of culture devices all crawl out stem cells, and the positive clone rate of the 96-well plate is only about 65.38 percent.
When the clones proliferate until passaging is possible, the cells are digested for counting and conventional culture. On day 6, the total tissue yield in the 96-well plate culture was 2.44×10 5/cm2, and the difference was up to 2-fold, although the culture vessel 100 was up to about 4.16×10 5/cm2. The cell morphology of both groups was also substantially similar during the culture.
After regular culture at approximately the same culture concentration (2.1×10 4/cm2～2.2*104/cm2), 3.77×10 7 cells were obtained on day 15 in the 100 groups of cultures, whereas the total cell yield in the 96-well plate group was 1.17×10 7, with a difference of up to 3 times.
In the primary culture experiments where the dental pulp is small in quantity and difficult to culture, the incubator 100 shows excellent performance, and the cloning number and the cell number of the culture are obviously superior to those of the conventional adherence control of 24-well plates/96-well plates. It is assumed that the culture vessel 1 of the incubator 100 is cultured in a fixed tissue, and that the ability to promote adhesion is the most important factor, and that the small-volume culture of the culture vessel 1 also has a high promoting effect on the proliferation of primary cells.
It can be seen that the culture tank 1 of the incubator 100 is a very excellent choice for primary culture with a small tissue amount, which is difficult to adhere like dental pulp. Especially for clinical puncture samples or other limited tissues, the success rate of primary culture is greatly improved.
While the preferred embodiments of the present invention have been described in detail above, it should be understood that aspects of the embodiments can be modified, if necessary, to employ aspects, features and concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above detailed description. In general, in the claims, the terms used should not be construed to be limited to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A culture device, the culture device comprising:
a culture cassette having an accommodation space and an opening at a top; and
The incubator, the incubator is made by polydimethylsiloxane and is located in accommodation space, the bottom surface of incubator is hugged closely the diapire of cultivateing the box, the lateral wall of incubator is hugged closely the inside wall of cultivateing the box, the incubator is equipped with:
a culture tank formed by recessing a bottom surface of the incubator;
An input hole formed by a top wall of the culture tank being recessed to a top surface of the incubator; and
And the output hole is formed from the top wall of the culture tank to the top surface of the incubator in a recessed mode and is arranged at intervals with the input hole.
2. A method of processing a culture vessel, comprising the steps of:
s1, mixing a silicon polymer and a cross-linking agent in proportion, and uniformly stirring to form a mixed colloid;
S2, extracting bubbles in the mixed glue;
S3, superposing a sheet-shaped mold on the bottom wall of the culture box, pouring the mixed colloid after air bubble extraction into the culture box, and heating and drying until the mixed colloid is solidified and molded to form a incubator;
S4, after the incubator is taken out, separating the die from the incubator, and forming a culture tank on the bottom surface of the incubator; and punching an input hole and an output hole on the top wall of the culture tank by using a puncher, and enabling the input hole and the output hole to penetrate through the culture tank respectively.
3. The method of claim 2, wherein the culture cassette is a petri dish and the mold is a slide.
4. The method according to claim 2, wherein in step S2, a plurality of the molds are stacked on the bottom wall of the culture cassette, respectively, and are arranged at intervals;
In step S4, a plurality of culture tanks are formed by separating the plurality of molds from the incubator, and the input holes and the output holes are punched in the top walls of the culture tanks by a puncher.
5. The method of processing an incubator according to claim 2, wherein at least two of the molds are stacked in a vertical direction.
6. The method of claim 5, wherein at least two of the molds are different in size.
7. The method of processing a culture vessel according to claim 2, wherein the heating temperature is 70 ° -80 ° for 60min-70min in step S3.
8. A method of cell culture comprising the steps of:
a. cleaning the culture device of claim 2 to remove impurities and residues on the surface of the culture device;
b. sterilizing the incubator and the culture box; c. placing the incubator within the incubator;
d. The culture medium is moved from the inlet port into the culture tank.
9. The cell culture method of claim 8, wherein the incubator is made of polydimethylsiloxane; in the step a, the incubator is placed in phosphate buffer salt solution for soaking for a preset time, impurities and residues on the surface of the incubator are removed, and then the incubator is placed in deionized water for ultrasonic cleaning, so that the cleanliness of the surface of the incubator is ensured.
10. The method of claim 9, wherein in step b, after sterilizing the incubator, the incubator is further wiped dry using a sterilizing filter paper.
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