Cell culture bottle capable of supporting repeated bottle laying of primary tissues and use method of cell culture bottle
By setting a partition and a nylon mesh filter layer inside the cell culture flask, the flask is divided into two spaces, which solves the problems of difficult tissue acquisition and low utilization rate in primary tissue cell culture, and realizes the reuse of tissue and efficient cell culture.
Patent Information
- Application Number
- CN202511021287.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-24
- Publication Date
- 2025-10-17
AI Technical Summary
In primary cell culture, tissue acquisition is difficult, few cells migrate out after plating, tissue fragments float up affecting plating, medium changes are difficult, and cells and tissues are difficult to separate, resulting in low utilization.
Design a cell culture flask with an internal partition and nylon mesh filter layer, dividing the flask into two spaces for primary culture and filtration of culture medium and cells. Use a TC-treated membrane to improve tissue utilization.
The design of the partition and nylon mesh filter layer enables repeated tissue placement in bottles, improves tissue utilization, simplifies cell harvesting and medium exchange processes, and enhances cell migration efficiency.
Smart Images
Figure CN120796066A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of cell culture technology, in particular to a cell culture flask for repeated plating of primary tissue and a method of using the same. BACKGROUND
[0002] Cell culture is one of the most commonly used means in biological and medical research, which can be divided into primary culture and subculture. Primary culture refers to taking cells, tissues or organs directly from a living organism and allowing them to maintain and grow in vitro. Since the cells of primary culture have just left the living organism, their biological characteristics have not changed much, and they are closer to the living state in the living organism. This method can provide a powerful means for studying the growth, metabolism and reproduction of cells in the living organism. Tissue block culture is a commonly used, simple and easy method with high success rate: after the tissue block is cut by mechanical method and attached to the cell culture flask, cell culture medium is added and placed in a carbon dioxide incubator for static culture. Cells can crawl out from the four sides of the tissue block within 1-2 weeks.
[0003] In the process of primary tissue cell culture, the tissue is complex and difficult to obtain, and the tissue is valuable. After plating once, the cells that crawl out are less, which cannot meet the needs of experiments. At the same time, after the tissue block is attached to the flask, the addition of cell culture medium can easily cause the tissue block to float, which affects the attachment of the tissue block and further affects the cell crawling rate. When changing the liquid in 5-7 days, due to the influence of the tissue block, the culture medium is not easy to suck out, which can easily cause the blockage of the pipette. When the cells crawl out, the tissue block and the cells are mixed and not easy to separate. SUMMARY
[0004] In order to solve the problems of the prior art, the present application provides a cell culture flask for repeated plating of primary tissue and a method of using the same. The cell culture flask is divided into two parts, and the tissue can be repeatedly plated, which greatly improves the utilization rate of the tissue.
[0005] The technical scheme adopted by the present application is as follows: A cell culture flask for repeated plating of primary tissue, comprising a flask body, a partition plate is arranged in the flask body, the upper edge of the partition plate reaches the bottle mouth, the lower edge of the partition plate is located in the middle and lower part of the flask body, and a nylon mesh filter layer is arranged between the lower edge of the partition plate and the inner wall of the flask body, the nylon mesh filter layer and the partition plate divide the inside of the flask body into a first culture space and a second culture space, the first culture space is used for primary culture of the repeated plating of primary tissue, and the second culture space is used for filtering out the culture medium after primary culture and the primary culture cells.
[0006] Preferably, the pore size of the nylon mesh filter layer is 100 um.
[0007] Preferably, the material of the partition plate is polystyrene.
[0008] A method for using a cell culture flask supporting repeated plating of primary tissue, comprising the following steps: A. cutting the primary tissue into pieces, plating into the first culture space of the cell culture flask, and placing in a cell culture incubator at 37°C, 5% CO2 for 4-6 hours; B. adding cell culture medium to the bottom of the cell culture flask through the bottle opening of the second culture space, then slowly placing the cell culture flask to make the cell culture medium slowly fill the entire first culture space, so that the primary tissue pieces are soaked in the cell culture medium; C. After 3-7 days, observe the cell outgrowth, place the cell culture flask vertically, so that the cell culture medium flows back to the bottom of the cell culture flask through the nylon mesh filter layer, and the primary tissue floating up is intercepted in the first culture space, then the used cell culture medium is half or full volume exchanged through the second culture space; D. After 14 days, when new cells grow out, harvest the cells: the cells are digested by trypsin and fall off from the inner wall of the culture flask, enter the bottom of the culture flask through the nylon mesh filter layer, and finally the regenerated cells without tissue fragments are obtained from the second culture space for subculture; E. After washing the tissue pieces left in the first culture space of the cell culture flask with physiological saline for 2-3 times, tear off a thin film, continue to plate the tissue pieces into the first culture space, and repeat steps B-D, to carry out the secondary culture.
[0009] Preferably, in step E, the thin film is treated with TC.
[0010] Preferably, the primary tissue is umbilical cord tissue.
[0011] Preferably, the umbilical cord tissue is pretreated by the following steps: A1. Place the umbilical cord in a 150 mm culture dish, wash the umbilical cord thoroughly with physiological saline for at least 3 times to remove surface blood clots and other substances, cut off 1 cm length from each end of the inside of the umbilical cord ligation port with a sharp straight scissors, and discard; wash the umbilical cord tissue at least 2 more times until the washing liquid becomes clear; A2. Cut the umbilical cord into 2 cm long pieces with a sharp straight scissors, wash 3 times to remove blood clots in the umbilical cord; transfer the umbilical cord tissue to a new culture dish, add physiological saline to the culture dish until the umbilical cord tissue is submerged by 1 / 2; make an incision on one end of the umbilical cord along the parallel direction of the vein, tear the vein edge longitudinally along the incision direction with a tissue forceps, and separate 1 vein and 2 artery blood vessels; remove the amnion with a tissue forceps and tear the Wharton's jelly; A3. Place the Wharton's jelly in a 50 ml centrifuge tube containing physiological saline, and cut it into 1 mm 3 ~ 3 mm 3 size with surgical scissors.
[0012] The technical scheme provided by the application has the beneficial effects that: The application divides the culture bottle into two parts by using a nylon net with a pore size of 100 um and a polystyrene partition plate, one part is a normal culture bottle, and the other part is used for liquid addition and replacement during cell culture. Several layers of thin films treated by TC are laid in the culture bottle. After the first harvest of tissues is completed, a layer of thin film is torn off to leak the next layer of thin film treated by TC. After the tissues are laid flat in the bottle and continue to be cultured, the utilization rate of the tissues can be greatly improved. BRIEF DESCRIPTION OF DRAWINGS
[0013] In order to more clearly illustrate the technical scheme in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.
[0014] Figure 1 It is a structure sectional view of a cell culture bottle supporting repeated plating of primary tissues. Figure 2 It is a use state diagram of a cell culture bottle supporting repeated plating of primary tissues. Figure 3 It is a use state diagram of a cell culture bottle supporting repeated plating of primary tissues. Figure 4 It is a use state diagram of a cell culture bottle supporting repeated plating of primary tissues. DETAILED DESCRIPTION
[0015] In order to make the purpose, technical scheme and advantages of the application more clear, the embodiments of the application will be further described in detail below with reference to the drawings. Embodiment one
[0016] The present embodiment is shown as follows. Figure 1 A cell culture bottle is provided, which includes a bottle body 1, a partition plate 2 is arranged inside the bottle body 1, the upper edge of the partition plate 2 reaches the bottle opening, the lower edge of the partition plate 2 is located in the middle lower part of the bottle body 1, and a nylon net filter layer 3 is arranged between the lower edge of the partition plate 2 and the inner wall of the bottle body, the nylon net filter layer 3 and the partition plate 2 divide the inside of the bottle body into a first culture space 4 and a second culture space 5, the first culture space 4 is used for primary culture of repeated plating of primary tissues, and the second culture space 5 is used for filtering out the culture medium after primary culture and the primary culture cells.
[0017] The pore size of the nylon mesh filter layer 3 of the present embodiment is 100 um. The material of the partition 3 is polystyrene. Example Two
[0018] The present embodiment is based on the structure of the cell culture flask of the embodiment, and provides a method for using a cell culture flask supporting repeated plating of primary tissues. The method divides the cell culture flask into two parts, which can improve the utilization rate of tissues by repeated plating of tissues.
[0019] The cell culture flask is divided into two parts by a nylon mesh with a pore size of 100 um and a polystyrene partition. One part is a normal culture flask, and the other part is used for liquid addition and replacement during cell culture. Several layers of TC-treated membranes are plated in the culture flask. After the first harvest of tissues is completed, one layer of membrane is torn off to leak the next layer of TC-treated membrane. After the tissues are plated in the flask, continue to culture, which can greatly improve the utilization rate of tissues.
[0020] As shown in the accompanying Figure 2 The primary tissues 6 are cut into fragments by a mechanical method and plated into the first culture space 4 of the cell culture flask, and then placed in a cell culture incubator (37°C, 5% CO2) for 4-6 hours. Then the cell culture medium 7 is added to the bottom of the cell culture flask through the second culture space 5. As shown in the accompanying Figure 3 Slowly place it flat so that the cell culture medium slowly fills the entire culture flask.
[0021] As shown in the accompanying Figure 4 After 3-7 days, observe the cell outgrowth, place the cell culture flask vertically, and allow the cell culture medium to flow smoothly through the nylon mesh filter layer part to the bottom of the cell culture flask. The floating tissues are intercepted in the first culture space, and the cell culture medium is replaced by half or full volume through the second culture space.
[0022] Repeat the above steps, and slowly place it flat again so that the cell culture medium slowly fills the entire culture flask. After the cells outgrow after about 14 days, harvest the cells. When the cells are harvested, the cells are detached from the culture flask by trypsin digestion, and pass through the nylon mesh filter layer to the bottom of the culture flask. Clean cells without tissue fragments are obtained from the second culture space for subculture.
[0023] After the tissues left in the first culture space of the cell culture flask are washed with normal saline for 2-3 times, one layer of membrane is torn off, and the tissues are plated in part 1. After adding the cell culture medium, the tissues are cultured again.
[0024] The use of the cell culture flask of the present embodiment is further illustrated below using an umbilical cord as an example. The method includes the following steps: 1. Put the umbilical cord into a 150mm culture dish, wash the umbilical cord with physiological saline for at least 3 times to remove the surface blood and other substances, cut off about 1cm length of the two ends of the umbilical cord inside the ligation port with a straight scissors with a sharp tip, and discard. Wash the umbilical cord tissue for at least 2 times until the washing liquid becomes clear.
[0025] 2. Cut the umbilical cord into small pieces of about 2cm in length with a straight scissors with a sharp tip, and wash the umbilical cord for 3 times to remove the blood. Transfer the umbilical cord tissue to a new culture dish, and add physiological saline to the culture dish to submerge the umbilical cord tissue by 1 / 2. Cut an incision on one end of the umbilical cord along the parallel direction of the venous blood vessels, and tear the venous blood vessels along the incision direction with a tissue forceps to separate 1 venous blood vessel and 2 arterial blood vessels. Remove the amnion with a tissue forceps, and tear the Wharton's jelly.
[0026] 3. Put the Wharton's jelly into a 50ml centrifuge tube with a small amount of physiological saline, and cut the Wharton's jelly into pieces of 1mm 3 ~3mm 3 in size with a surgical scissors.
[0027] 4. Spread the Wharton's jelly pieces evenly into the first culture space of a cell culture bottle, and place the cell culture bottle into a cell culture box (37℃, 5% CO2) for 4 hours. Then, add an appropriate amount of cell culture medium into the second culture space of the cell culture bottle, and place the cell culture bottle horizontally to slowly fill the whole cell culture bottle with the cell culture medium to cover the tissue. Then, place the cell culture bottle into the cell culture box (37℃, 5% CO2) for culture.
[0028] 5. After 7 days, a small amount of cells are observed to grow out of the tissue, and the cell culture medium is removed from the second culture space with a pipette. Then, new cell culture medium is added into the second culture space.
[0029] 6. After 14 days, the cells are harvested by trypsin digestion, and the cells are collected from the bottom of the culture bottle through a nylon net. Then, the cells without tissue fragments are obtained from the second culture space for subculture.
[0030] The tissue in the first culture space is washed with physiological saline for 3 times, and the upper film is torn off with a high-temperature sterilized forceps to leak the lower film treated with TC. Then, the tissue is cultured again, and the steps 5 to 6 are repeated to improve the utilization rate of the precious tissue.
[0031] The above only describes the preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A cell culture flask supporting repeated plating of primary tissues, comprising a flask body, characterized in that: A partition is provided inside the bottle body, the upper edge of the partition reaches the bottle mouth, the lower edge of the partition is located in the lower middle part of the bottle body, and a nylon mesh filter layer is provided between the lower edge of the partition and the inner wall of the bottle body. The nylon mesh filter layer and the partition divide the inner part of the bottle body into a first culture space and a second culture space. The first culture space is used for repeated bottling of primary tissue for primary culture, and the second culture space is used to filter out the culture medium and primary culture cells after the primary culture.
2. A cell culture flask supporting repeated plating of primary tissue according to claim 1, characterized in that: The pore size of the nylon mesh filter layer is 100 μm.
3. The cell culture flask supporting repeated plating of primary tissue according to claim 1, characterized in that: The material of the partition is polystyrene.
4. A method for using the cell culture flask supporting repeated plating of primary tissue according to any one of claims 1 to 3, comprising the following steps: A. Cut the primary tissue into pieces and spread them into the first culture space of the cell culture flask. Place the pieces in a cell culture incubator at 37°C and 5% CO2 for 4-6 hours. B. Add the cell culture medium to the bottom of the cell culture flask through the mouth of the second culture space, then slowly level the cell culture flask so that the cell culture medium slowly fills the entire first culture space, so that the primary tissue fragments are immersed in the cell culture medium; C. After 3-7 days, observe the cells crawling out. Place the cell culture flask upright to allow the cell culture medium to flow back to the bottom of the cell culture flask through the nylon mesh filter layer. The floating primary tissue is intercepted in the first culture space. Then, the cell culture medium used in the culture is replaced with half or all of the medium in the second culture space. D. After 14 days, when the new cells have crawled out, the cells are harvested. During cell harvesting, the cells are digested with trypsin and fall off the inner wall of the culture flask. They then pass through the nylon mesh filter layer and enter the bottom of the culture flask. Finally, clean regenerative cells without tissue fragments can be obtained from the second culture space for subculture. E. Wash the tissue fragments remaining in the first culture space of the cell culture flask with physiological saline 2-3 times, tear off a layer of film, and continue to spread the tissue fragments into the first culture space. Repeat steps B to C for re-culture.
5. The method for using a cell culture flask supporting repeated plating of primary tissue according to claim 4, characterized in that: In the step E, the film is subjected to TC treatment.
6. The method for using a cell culture flask supporting repeated plating of primary tissue according to claim 4, characterized in that: The primary tissue is umbilical cord tissue.
7. The method for using a cell culture flask supporting repeated plating of primary tissue according to claim 4, characterized in that: The umbilical cord tissue is pretreated by the following steps: A1. Place the umbilical cord in a 150mm culture dish and thoroughly wash it at least three times with normal saline to remove any blood or other debris from the surface. Use pointed straight scissors to cut off 1 cm from each end of the inner side of the ligature and discard. Wash the umbilical cord tissue at least two more times until the wash solution becomes clear. A2. Cut the umbilical cord into 2 cm long pieces using pointed straight scissors. Rinse the cord three times to remove any blood clots. Transfer the umbilical cord tissue to a new culture dish and add saline to cover half of the cord tissue. Make an incision at one end of the umbilical cord parallel to the vein. Use forceps to tear the vein longitudinally along the incision to separate one vein and two arteries. Use forceps to remove the amniotic membrane and Wharton's jelly. A3. Place Wharton's jelly in a 50ml centrifuge tube with normal saline and cut it into 1mm pieces with surgical scissors. 3 ~3mm 3 size.