CN112266875B

CN112266875B - Cell culture container and method for culturing primary cells

Info

Publication number: CN112266875B
Application number: CN202011022153.8A
Authority: CN
Inventors: 徐洪杰; 秦大江
Original assignee: Bioisland Laboratory
Current assignee: Bioisland Laboratory
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2021-10-29
Anticipated expiration: 2040-09-25
Also published as: CN112266875A

Abstract

The invention relates to a cell culture container and a method for culturing primary cells, wherein the cell culture container comprises at least one culture unit, the culture unit comprises a culture tank, a fixing piece and a cover body, the culture tank is used for containing cell culture solution, the fixing piece is positioned on the tank bottom of the culture tank and is used for fixing a tissue block so as to enable the tissue block to be attached to the tank bottom of the culture tank, a cover plate can be contained in the culture tank, and the cover plate is movably connected with the fixing piece. The cell culture container can improve the cell yield of primary culture.

Description

Cell culture container and method for culturing primary cells

Technical Field

The invention relates to the technical field of cell culture, in particular to a cell culture container and a method for culturing primary cells.

Background

Primary culture refers to the first culture, also called primary culture, in which tissues or cells are removed from the body. The primary culture has short in vitro time and similar genetic characters to in vivo cells, and is suitable for cell morphology, function, differentiation and other researches. The most commonly used primary culture is tissue mass culture and dispersed cell culture. The tissue mass culture method is to transplant the cut tissue mass directly into a culture vessel, and then culture the tissue mass after adding a culture medium. The dispersed cell culture method comprises digesting intercellular conjugates with proteolytic enzyme (such as trypsin and collagenase), or removing Ca dependent on cell adhesion with metal ion chelating agent (such as EDTA)²⁺Then, the cells are mechanically shaken slightly to form single cells, and then the cells are cultured after a culture medium is added.

In clinical application, the tissue mass culture method has higher safety because cells are not treated by heterologous proteolytic enzyme, however, the yield of primary cells is lower when the tissue mass culture method is adopted for primary culture.

Disclosure of Invention

In view of this, there is a need for a cultured cell container that can improve the yield of cells in primary culture.

Further, a method of culturing primary cells capable of increasing the cell yield of the primary culture is also provided.

A cell culture vessel comprising at least one culture unit, the culture unit comprising:

a culture tank for containing a cell culture solution;

the fixing piece is fixed on the bottom of the culture tank and used for fixing the tissue block so that the tissue block is attached to the bottom of the culture tank; and

the cover plate can be contained in the culture tank and is movably connected with the fixing piece.

Above-mentioned cell culture container is fixed in the tank bottom of culture tank with the tissue piece through the mounting for the tissue piece pastes the tank bottom to the culture tank more easily, reduces the influence of culture solution to the tissue piece adherence, and through the suppression with mounting swing joint's apron, has further improved the adherence rate of tissue piece, promotes to paste to the tank bottom growth of culture tank from the cell that the cell climbed out, and then improves primary culture's cell output.

In one embodiment, the number of the fixing parts is multiple, and the fixing parts are fixed on the bottom of the culture tank in parallel and at intervals.

In one embodiment, the fixing member has a connecting portion and a puncturing portion connected to the connecting portion, the connecting portion is fixedly connected to the bottom of the culture tank, the puncturing portion extends in a direction away from the bottom of the culture tank, and the cross-sectional area of the puncturing portion gradually decreases from an end close to the connecting portion to an end away from the connecting portion.

In one embodiment, the fixing member is cylindrical, the length of the fixing member is less than the depth of the culture tank, the length of the fixing member is 5mm to 20mm, and the diameter of the fixing member is 1mm to 2 mm.

In one embodiment, the cover plate is provided with a fixing hole, the cover plate is sleeved on the fixing piece through the fixing hole, and the orthographic projection area of the fixing hole on the bottom of the culture tank is smaller than the area of the surface of the tissue block, which is in contact with the cover plate.

In one embodiment, the cover plate is provided with a vent hole, and the vent hole is close to the center of the cover plate.

In one embodiment, the cover plate is a glass cover plate or a plastic cover plate.

In one embodiment, the culture unit is provided in plurality, and a plurality of the culture units are arranged at intervals.

In one embodiment, the cell culture container further comprises a cover for covering the notch of the culture tank.

A method of culturing primary cells comprising the steps of:

fixing the tissue block on the fixing part of the cell culture container, and attaching one surface of the tissue block to the bottom of a culture groove of the cell culture container;

sleeving a cover body of the cell culture container on the fixing piece, wherein the cover body is pressed on the tissue block; and

adding a culture solution to the cell culture vessel.

Drawings

FIG. 1 is a cell culture vessel according to an embodiment;

FIG. 2 is a cover plate of another embodiment;

FIG. 3 is a plan view of another embodiment of a cell culture vessel.

Reference numerals: 10. a cell culture vessel; 100. a culture unit; 110. a culture tank; 120. a fixing member; 130. a cover plate; 131. a fixing hole; 132. and (4) air holes.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Referring to fig. 1, according to one embodiment of the present invention, a cell culture container 10 is provided, in which a culture unit 100 is disposed on the cell culture container 10, and the culture unit 100 includes a culture tank 110, a fixing member 120, and a cover plate 130.

Specifically, the culture tank 110 is used for holding a cell culture solution, and provides a place for the cells to climb out of and grow in the tissue mass. In one embodiment, the material of the culture tank 110 is plastic (e.g., polystyrene) or glass. Of course, in other embodiments, the material of the culture tank 110 may be other materials as long as the cells are not prevented from climbing out of the tissue mass.

In the embodiment shown in FIG. 1, the culture tank 110 has a cylindrical shape. Of course, in other embodiments, the shape of the culture tank 110 is not limited to a cylindrical shape, but may be other shapes such as a cone shape, a square shape, and the like.

In one embodiment, the bottom of the culture tank 110 is a flat bottom. In another embodiment, the bottom of the culture tank 110 is rounded.

In one embodiment, the culture tank 110 is cylindrical, and the diameter of the opening of the culture tank 110 is 35 mm-150 mm; the depth of the culture tank 110 is 10mm to 25 mm. Further, the diameter of the opening of the culture tank 110 is 60 mm-150 mm; the depth of the culture tank 110 is 10mm to 20 mm. Of course, in other embodiments, the size of the culture tank 110 is not limited to the above, and may be adjusted according to more practical requirements (e.g., culture amount).

The fixing member 120 is fixed on the bottom of the culture tank 110 and is used for fixing the tissue block, so that the tissue block is attached to the bottom of the culture tank 110, and cells climbing out of the tissue block can adhere to the wall. Specifically, the fixing member 120 has a cylindrical shape, such as a cylinder, a triangular prism, a quadrangular prism, or the like. Further, the fixing member 120 has a barb shape or a barbed shape. The fastener 120 is configured to be barb-like or barbed to facilitate fixation of the tissue mass. In the embodiment shown in fig. 1, the fixing member 120 has a cylindrical shape. Further, the fixing member 120 has a connecting portion connected to the bottom of the culture tank 110 and a piercing portion connected to the connecting portion, the piercing portion extends in a direction away from the bottom of the culture tank 110, and the cross-sectional area of the piercing portion gradually decreases from one end close to the connecting portion to one end away from the connecting portion. The piercing part is arranged as above, so that the fixing part 120 can pierce the tissue block conveniently, and the tissue block can be attached to the bottom of the groove conveniently. In an alternative specific example, the piercing portion is tapered.

Specifically, the number of the fixing members 120 is plural, and the plurality of fixing members 120 are provided at intervals on the bottom of the culture tank 110. In the embodiment shown in FIG. 1, there are 9 fixing members 120, and 9 fixing members 120 are arranged in a matrix at the bottom of the culture tank 110. Of course, in other embodiments, the number of the fixing members 120 is not limited to 9, and may be any other integer larger than 0. Such as 2, 4, 8, 12, etc. Of course, in some embodiments, there may be one fixing member 120.

In one embodiment, the fixing member 120 is integrally formed with the culture tank 110. The fixing member 120 is integrally formed with the culture tank 110 to facilitate cleaning of the culture tank 110, and to prevent the fixing member 120 from being difficult to clean due to a gap between the fixing member and the bottom of the culture tank 110.

The cover plate 130 is movably connected to the fixing member 120 and can be received in the culture tank 110. During the culture process, the cover plate 130 is used for pressing tissue blocks, so that the floating or drifting of the tissue blocks is reduced, and the adherence rate of the tissue blocks is improved.

Specifically, the cover plate 130 is provided with a fixing hole 131, and the cover plate 130 can be sleeved on the fixing member 120 through the fixing hole 131. It is understood that the area of the orthographic projection of the fixing hole 131 on the bottom of the culture tank 110 is smaller than the area of the surface of the tissue mass in contact with the cover plate 130 to prevent the cover plate 130 from directly sliding down to the bottom of the culture tank 110 without pressing the tissue mass. In the embodiment shown in fig. 1, the cover plate 130 has 4 circular fixing holes 131, 4 fixing holes 131 are arranged at intervals, and the 4 fixing holes 131 are located at four vertexes of a rectangle. Of course, in other embodiments, the number of the fixing holes 131 is not limited to 4, and may be any other integer greater than 0, and the shape of the fixing holes 131 is not limited to a circle, and may be other shapes, such as a square, a triangle, and the like.

In one embodiment, the fixing member 120 has a cylindrical shape, and the diameter of the fixing member 120 is 1mm to 2 mm; the length of the fixing member 120 is 1mm to 20 mm. In an alternative embodiment, the length of the fixing member 120 is 1mm, 3mm, 5mm, 8mm, 10mm, 15mm, 18mm or 20 mm. Further, the length of the fixing member 120 is 5mm to 20 mm. At this time, the diameter of the fixing hole 131 is matched with the diameter of the fixing member 120, and is 1.1mm to 2.1 mm. Of course, the length of the fixing member 120 is smaller than the depth of the culture tank 110. It is understood that in other embodiments, the sizes of the fixing member 120 and the fixing hole 131 are not limited to the above, and may be adjusted according to actual requirements.

Further, referring to fig. 2, the cover plate 130 is further provided with air holes 132. The vent holes 132 are provided to accelerate the sinking of the cover plate 130 through the vent holes 132 when the culture solution is added to the culture tank 110 having the tissue mass, and to remove air bubbles generated by the addition of the culture solution. In the embodiment shown in fig. 2, the airing hole 132 is near the center of the cover plate 130. In the embodiment shown in fig. 2, the fixing holes 131 are round holes, the number of the fixing holes 131 is 4, the number of the ventilation holes 132 is 8, the connection line of the 4 fixing holes 131 is square, and the ventilation holes 132 are distributed on the whole cover plate 130 at intervals.

It is understood that in some embodiments, the number of the cover plate 130 is plural. In use, the culture tank 110 can accommodate a plurality of cover plates 130. Of course, when the number of the cover plates 130 is plural, plural cover plates 130 may be stacked and laid.

In the present embodiment, the cover plate 130 is a plastic cover plate, for example, a polystyrene cover plate. Of course, in other embodiments, the cover plate 130 is not limited to a plastic cover plate, but may be a cover plate 130 of other materials, such as a glass cover plate.

In an alternative specific example, the cover plate 130 is circular, and the diameter of the cover plate 130 is 20mm to 100 mm. The air holes 132 are round holes, and the diameter of the air holes 132 is 0.2 mm-0.8 mm. Of course, in other embodiments, the shapes and sizes of the cover plate 130 and the ventilation holes 132 are not limited to the above, and may be adjusted according to actual situations.

It is understood that in some embodiments, the cover plate 130 may be omitted.

In some embodiments, cell culture vessel 10 further comprises a lid for covering the slot of culture tank 110. In an alternative specific example, the cover is flat. In another optional specific example, the cover body comprises a shielding portion and a limiting portion connected with an edge of the shielding portion. When in use, the shielding part is used for shielding the notch of the culture tank 110; spacing portion is used for restricting the lid, controls and rocks, avoids the lid landing. Furthermore, spacing portion includes many spacing posts, restricts the displacement range of lid through many spacing posts. Or the limiting part is a hollow structure with two open ends, and one end of the limiting part is fixedly connected with the shielding part.

It is understood that the cell culture vessel 10 is not particularly limited to the tissue mass to be cultured, and may be, for example, an umbilical cord tissue or a skin tissue, and is suitable for culturing primary cells by a tissue mass culture method.

The cell culture vessel 10 described above has at least the following advantages:

(1) the tissue block is fixed at the bottom of the culture tank 110 by the fixing part 120, so that the adherence rate of the tissue block is improved, the cells climbing out from the cells are promoted to adhere to the bottom of the culture tank 110 for growth, and the cell yield of primary culture is improved.

(2) By arranging the cover plate 130, the adherence rate of the tissue blocks is further improved, and the cell yield of the primary culture is further improved.

(3) Through setting up bleeder vent 132, be convenient for the tissue piece adherence, also be convenient for eliminate the bubble, do benefit to cell growth.

Referring to FIG. 3, another embodiment of a cell culture container 20 is provided, wherein the cell culture container 20 has a plurality of culture units 200, and the plurality of culture units 200 are disposed at intervals. In the embodiment shown in FIG. 3, 6 culture units 200 are provided at intervals in the cell culture vessel 20. By arranging a plurality of culture units 200 on one cell culture container 20, the same batch operation is facilitated, and the scale production is facilitated.

An embodiment of the present invention further provides a method for culturing primary cells, including the following steps: fixing the tissue mass to the fixing member of the cell culture container according to any one of the above embodiments, and attaching one surface of the tissue mass to the bottom of the culture vessel of the cell culture container; sleeving a cover body of the cell culture container on the fixing piece, wherein the cover body is pressed on the tissue block; and adding a culture medium to the cell culture vessel.

In an alternative embodiment, there are four fasteners and the tissue mass is in the form of a square. At this time, the tissue block is inserted into the fixing pieces, four corners of the tissue block correspond to the four fixing pieces, and one surface of the tissue block is attached to the bottom of the culture tank. Of course, in other embodiments, the number of the fasteners may be varied to fix the tissue mass to the fasteners and to smoothly cover the bottom of the culture tank.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

The structure of the cell culture vessel used for primary culture in example 1 is shown in FIG. 1. The cell culture container in example 1 had one culture unit, the culture tank was made of polystyrene, the diameter of the culture tank was 100mm, the depth of the culture tank was 20mm, the length of the fixing member was 10mm, the diameter of the fixing member was 1mm, the diameter of the lid plate was 92mm, the diameter of the vent hole was 0.6mm, and the diameter of the fixing hole was 1.1 mm.

The procedure of primary culture of example 1 is specifically as follows:

(1) collecting the umbilical cord: umbilical cords (1.5 cm in diameter) of a full-term healthy fetus produced by caesarean section were collected, immersed in PBS containing 1% (m/v) penicillin and 1% (m/v) streptomycin, and placed on ice.

(2) Tissue isolation: cutting the umbilical cord into 4 cm-long segments in a super clean bench, longitudinally splitting the segments respectively, repeatedly flushing the segments with sterile PBS until the liquid is free from blood contamination, removing one vein blood vessel and two artery blood vessels (three in all) with hemostatic forceps and ophthalmology, and separating to obtain the Huatong glue tissue.

(3) Shearing the tissue of the Huatong glue obtained in the step (2) to be less than 2mm³And the tissue block is drained after being washed by PBS, the tissue block is fixed on a fixing piece of the cell culture container, and then the cover plate is sleeved on the fixing piece fixed with the tissue block so that the tissue block is pressed by the cover plate. After 30 minutes in the incubator, DMEM/F12 medium containing 15% fetal bovine serum was added thereto at 37 ℃ with 5% CO₂Culturing in an incubator.

(4) The cell climbing-out around the tissue mass was observed under an inverted phase contrast microscope, and after 7 days, the medium was changed for the first time (the medium was changed by removing the culture supernatant from the dish, adding DMEM/F12 medium containing 15% (v/v) fetal bovine serum), and then every 4 days. When the cell expansion area was 80% of the area of the bottom of the dish, the cells were detached by digestion with 0.05% trypsin-EDTA and counted. Then subcultured into conventional petri dishes (polystyrene petri dishes without fixtures and cover plates, for cell culture) (first passage 1:2, followed by 1:3 or 1:4 passages every 3 days). Herein, a cell that climbed out of a tissue mass without passage is referred to as a zero-generation mesenchymal stem cell (P0), a mesenchymal stem cell obtained by subculturing the zero-generation mesenchymal stem cell is referred to as a first-generation mesenchymal stem cell (P1), a mesenchymal stem cell obtained by subculturing the first-generation mesenchymal stem cell is referred to as a second-generation mesenchymal stem cell (P2), and the like.

Statistically, in example 1, the amount of cells that climbed out per 4cm of umbilical cord tissue was 9.77X 10⁷The tissue block had an adherence rate (adherence rate ═ area of adherent tissue block/area of tissue block inoculated, the same applies hereinafter) of 82.2%.

(5) Performing phenotype identification on the third-generation mesenchymal stem cells: specifically, 1 × 10 is taken⁶For each cell, 100. mu.L of cell staining buffer was added. The corresponding volume of antibody [ mouse anti-human PE, APC or FITC labeled monoclonal antibodies CD105, CD44, CD90, CD29, CD34, CD45 and Human Leukocyte Antigen (HLA) -DR ] was then added according to the antibody instructions]Incubating at room temperature for 30min, centrifuging at 1000r/min for 5min, discarding the supernatant, and adding 500 μ L PBS to flow cytometry for detection. The results are shown in Table 1。

TABLE 1

As can be seen from Table 1, the positive rates of the surface expression of CD105, CD73, CD90, CD45, CD34, CD14, CD19 and HLA-DR of the third generation mesenchymal stem cells of example 1 meet the international standard (the cell surface of the mesenchymal stem cells express CD105, CD73 and CD90 (95% or more), and do not express CD45, CD34, CD14 or CD11b, CD79 alpha or (2%) as specified in the international standard).

Example 2

The primary culture procedure of example 2 is substantially the same as in example 1, except that the tissue used in example 2 is amniotic membrane tissue during the culture.

Statistically, in example 2, the amount of cells that climbed out per 4cm of umbilical cord tissue was 6.1X 10⁷The anchorage rate of the tissue mass was 79.9%.

The results of flow cytometry identification of the third generation mesenchymal stem cells in example 2 are shown in table 2.

TABLE 2

As can be seen from Table 2, the positive rates of the surface expression of CD105, CD73, CD90, CD45, CD34, CD14, CD19 and HLA-DR of the third generation mesenchymal stem cells of example 2 meet the international standard (the cell surface of the mesenchymal stem cells express CD105, CD73 and CD90 (95% or more), and do not express CD45, CD34, CD14 or CD11b, CD79 alpha or (2%) as specified in the international standard).

Comparative example 1

The primary culture procedure of comparative example 1 is substantially the same as in example 1, except that the vessel holding the tissue mass in comparative example 1 is a conventional culture dish (without fixture and cover plate) for culturing cells during the culture process.

As a result of statistics, in comparative example 1, the amount of cells that climbed out per 4cm of umbilical cord tissue was 1.01X 10⁷The anchorage rate of the tissue mass was 9.1%.

The results of flow cytometry identification of the third generation mesenchymal stem cells in comparative example 1 are shown in table 3.

TABLE 3

As can be seen from Table 3, the positive rates of the surface expression of CD105, CD73, CD90, CD45, CD34, CD14, CD19 and HLA-DR of the third generation mesenchymal stem cell of comparative example 1 meet the international standard (the international standard specifies that the cell surface of the mesenchymal stem cell expresses CD105, CD73 and CD90 (95%) but does not express CD45, CD34, CD14 or CD11b, CD79 alpha or (2%)).

Comparative example 2

The primary culture procedure of comparative example 2 is substantially the same as in example 2, except that the vessel holding the tissue mass in comparative example 2 is a conventional culture dish (without fixture and cover plate) for culturing cells during the culture process.

As a result of statistics, in comparative example 2, the amount of cells that climbed out per 4cm of umbilical cord tissue was 0.68X 10⁷The anchorage rate of the tissue block was 7.8%.

The results of flow cytometry identification of the third generation mesenchymal stem cells in comparative example 2 are shown in table 4.

TABLE 4

As can be seen from Table 4, the positive rates of the surface expression of CD105, CD73, CD90, CD45, CD34, CD14, CD19 and HLA-DR of the third generation mesenchymal stem cell of comparative example 2 meet the international standard (the cell surface of the mesenchymal stem cell expresses CD105, CD73 and CD90 (95%) and does not express CD45, CD34, CD14 or CD11b, CD79 alpha or (2%)).

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A cell culture vessel for primary culture of mesenchymal stem cells, comprising at least one culture unit comprising:

a culture tank for containing a cell culture solution;

the fixing pieces are fixed on the bottom of the culture tank in a mutually parallel and spaced mode, the fixing pieces are used for fixing tissue blocks so that the tissue blocks can be attached to the bottom of the culture tank, the fixing pieces are cylindrical, the diameter of each fixing piece is 1-2 mm, the length of each fixing piece is 1-20 mm, each fixing piece is provided with a connecting portion and a puncturing portion connected with the connecting portion, the connecting portions are fixedly connected with the bottom of the culture tank, the puncturing portions extend in the direction far away from the bottom of the culture tank, the cross sectional area of each puncturing portion is gradually reduced from one end close to the connecting portions to one end far away from the connecting portions, and the length of each fixing piece is smaller than the depth of the culture tank; and

the cover plates can be flatly accommodated in the culture tank and are movably connected with the fixing piece, the cover plates are circular, the diameter of each cover plate is 20 mm-100 mm, a plurality of air holes are formed in the cover plates and are close to the centers of the cover plates, the air holes are round holes, and the diameter of each air hole is 0.2 mm-0.8 mm; the cover plate is provided with a plurality of fixing holes, the cover plate is sleeved on the fixing part through the fixing holes, the area of the orthographic projection of the fixing holes on the bottom of the culture tank is smaller than the area of the surface of the tissue block, which is in contact with the cover plate, and the cover plate is a glass cover plate or a plastic cover plate.

2. The vessel for cell culture according to claim 1, wherein the culture unit is provided in plurality, and the plurality of culture units are arranged at intervals.

3. A primary culture method of mesenchymal stem cells is characterized by comprising the following steps:

fixing a tissue mass to the fixture of the cell culture vessel according to any one of claims 1 to 2, and attaching one surface of the tissue mass to the bottom of a culture vessel of the cell culture vessel;

sleeving a cover plate of the cell culture container on the fixing piece, wherein the cover plate is pressed on the tissue block; and

adding a culture solution to the cell culture vessel.