Search

CN-116286357-B - 3D printing organoid culture bracket and organoid culture method

CN116286357BCN 116286357 BCN116286357 BCN 116286357BCN-116286357-B

Abstract

The invention discloses a 3D printing organoid culture support and organoid culture method, which are characterized by comprising a main body, wherein a micro-channel is arranged in the main body, one end of the main body is provided with an inlet, the other end of the main body is provided with an outlet, the inlet and the outlet are respectively communicated with the micro-channel, the main body comprises a main branch and a support, the micro-channel is arranged in the support, the micro-channel is communicated with the inlet and the outlet through the main branch, a plurality of groups of micropores are arranged on the outer wall of the support at intervals, the micropores are communicated with the micro-channel, the support is formed by interconnecting a plurality of groups of branches, a plurality of groups of culture holes communicated with each other are formed between adjacent branches, micro-channels extending along the branches are arranged in each group of branches, the micro-channels in the plurality of groups of branches are communicated with each other, the micropores are arranged on the outer wall of the branches at intervals, and the micropores are communicated with the culture holes. The invention improves the culture effect of organoids.

Inventors

  • HAN KUNYUAN
  • HAN QINGLIN
  • LI YUHAI

Assignees

  • 润原生物科技(苏州)有限公司

Dates

Publication Date
20260512
Application Date
20230328

Claims (10)

  1. 1. The 3D printing organoid culture bracket is characterized by comprising a main body, wherein a micro-channel is arranged in the main body, one end of the main body is provided with an inlet, the other end of the main body is provided with an outlet, and the inlet and the outlet are respectively communicated with the micro-channel; The main body comprises two groups of main branches and a bracket, the micro-channels are arranged in the bracket, two ends of the bracket are respectively connected with the inlet and the outlet through the two groups of main branches, and the micro-channels are communicated with the inlet and the outlet through the main branches; a plurality of groups of micropores are arranged on the outer wall of the bracket at intervals, and the micropores are communicated with the micro-channels; The support is formed by connecting a plurality of groups of branches, a plurality of groups of mutually communicated culture holes are formed between adjacent branches, and the culture holes and the support provide a 3D growth microenvironment with a support structure for cells; The support comprises a plurality of groups of plane frames and a plurality of groups of vertical frames, the plurality of groups of plane frames are arranged at intervals, and each group of vertical frames connect adjacent plane frames; The culture holes are formed between the adjacent plane frames and the adjacent vertical frames; The interior of each group of the branches is provided with micro-channels extending along the branches, and the micro-channels in the groups of the branches are mutually communicated; The micro-holes are arranged on the outer wall of the branch at intervals, the micro-holes are used for communicating the micro-channels with the culture holes, the micro-holes provide culture medium for cells in the culture holes, and the culture medium provides nutrition and material exchange for the cells.
  2. 2. The 3D printing organoid culture support of claim 1, wherein a main runner is arranged in the main limb, and the micro runner is respectively communicated with the inlet and the outlet through the main runner.
  3. 3. The 3D printing organoid culture support according to claim 1, wherein an outer sleeve is further arranged outside the support, a cavity is formed in the outer sleeve, the outer end of the support is connected with the inner wall of the cavity, and the main limbs are arranged at two ends of the outer sleeve; the outer wall of the outer sleeve is provided with a plurality of groups of through holes communicated with the cavity, and the diameter of each through hole is larger than the outer diameter of each branch.
  4. 4. The 3D printing organoid culture support of claim 3, wherein end micropores are formed in the limbs of the joint of the support and the outer sleeve, and the end micropores are communicated with the outer surface of the outer sleeve.
  5. 5. The 3D printed organoid culture scaffold of claim 1, wherein: the branches comprise a first branch, a second branch and a third branch; The plane frame comprises a plurality of groups of first branches and second branches which are mutually connected in a cross mode, micro-channels are arranged in each group of the first branches and the second branches, and the micro-channels in the first branches are communicated with the micro-channels in the second branches; The vertical frame comprises a plurality of groups of third branches, the ends of the third branches are connected with the plane frame, and the micro-channels in the third branches are communicated with the micro-channels in the first branches and/or the second branches.
  6. 6. The 3D printing organoid culture support of claim 1, wherein the diameter of the culture hole is 100-1000 microns, the diameter of the micro-channel is 2-500 microns, and the diameter of the micro-hole is smaller than or equal to the diameter of the micro-channel.
  7. 7. The 3D printed organoid culture scaffold of claim 3, wherein the outer sheath is of a spherical or spheroid-like structure.
  8. 8. The 3D printing organoid culture support of claim 1, wherein the support is a planar support, the planar support comprises a plurality of groups of branches which are arranged at intervals from top to bottom, two ends of each group of branches are respectively connected with the main branches at two sides, and micro-channels in the branches are respectively communicated with an inlet and an outlet through the main branches at two sides.
  9. 9. The method for cultivating the organoid is characterized by comprising the following steps: ① Printing the 3D printed organoid culture scaffold of any of claims 1-8 with a 3D printer; ② Placing the culture support prepared in the step ① into a buffer solution for full swelling, and then placing the culture support into a chamber of a microfluidic chip; ③ Dripping cells and a culture medium to be cultured onto the culture bracket by using a dropper through a feeding port of the microfluidic chip, so that the culture bracket is fully immersed by the culture medium; ④ Placing the micro-fluidic chip with the culture bracket and the cells into a carbon dioxide incubator for culture, and conveying and replacing a culture medium and a growth promoting substance in the micro-fluidic chip by utilizing a peristaltic pump; In the step ④, an inlet of the culture support is connected with an inlet of the microfluidic chip, an outlet of the culture support is connected with an outlet of the microfluidic chip, and the microfluidic chip fixes the culture support in the cavity; The culture medium flows into the inlet of the culture bracket through the inlet of the microfluidic chip, is further sent to any position of the culture bracket through the micro-channel and the micro-holes for cell absorption culture, and flows out of the microfluidic chip through the outlet of the culture bracket, so that the substance exchange of the culture bracket is realized.
  10. 10. The method according to claim 9, wherein in the step ①, the process for producing the culture scaffold is as follows: a. printing and forming the culture support integrally by using a 3D printer to obtain a preformed culture support; b. And cleaning, drying and sterilizing the preformed culture bracket in sequence to obtain the formed culture bracket.

Description

3D printing organoid culture bracket and organoid culture method Technical Field The invention relates to the field of organoid culture, in particular to a 3D printing organoid culture bracket and a organoid culture method. Background In the last decade, great progress has been made in the field of stem cell research, with a major breakthrough being the rapid evolution of organoid (Organoids) systems from scratch. Organoid culture belongs to the advanced stage of 3D cell culture and is mainly characterized in that cell culture differentiation generates cell groups with different functions, and the organoid culture can simulate the related functions of human organs to a certain extent. The head and foot can be developed in the medical field, such as tumor drug sensitivity detection, high flux drug screening and the like, and has wide research and application potential. The final goal of organoid development is to culture human organs in vitro, thereby being capable of replacing the market of allograft transplantation and having great medical value and market prospect. However, to date, organoids have "died" to some extent, and current research has progressed far beyond the extent of organ culture in vitro. In short, the cells generate necrosis centers (necrotic core) due to continuous differentiation and expansion in the growth process, and the growth promoting substances such as culture medium and the like can not penetrate into the organoid centers to exchange substances, and the vascular network covering the whole body is arranged in the human body, so that various tissues and the inside can be supplied with nutrition and discharged waste, and the physiological functions of the human body are well maintained. The current internationally mainstream organoid culture mainly comprises two culture modes of conventional culture plate culture and high-flux micro-flow control (namely organoid chip), wherein the conventional culture plate culture generally adopts matrigel and special culture medium loaded with growth factors to culture cells, the culture mode needs to manually and periodically replace the culture medium, the high-flux micro-flow control generally implants the cells in a chamber (chamber) in the middle of a micro-flow control flow channel, and the culture medium loaded with substances required by organoid growth differentiation is pumped into the chamber for cell growth in the micro-flow control chip through a peristaltic pump through a single or a plurality of inlets and outlets, so that the automatic replacement of the culture medium is realized. In addition to Matrigel, microspheres and other hydrogel materials can also be used in the chamber to create a 3D microenvironment that provides support for cell growth. In either of the above-mentioned ways, there is a great difficulty that the cells in the center are less than the medium (i.e., material exchange) obtained by the surface cells during the process of differentiating into the organoids, resulting in cells that grow to a certain extent to be withered from the center, and finally, organoids are difficult to continue to grow after differentiating to a certain extent, thus failing to further simulate the relevant functions of human organs. Disclosure of Invention The invention aims to provide a 3D printing organoid culture bracket and a organoid culture method, by using the structure and the method, the contact amount of cells in the center and a culture medium can be increased in the cell culture process, and the culture effect is further improved. In order to achieve the aim, the technical scheme adopted by the invention is that the 3D printing organoid culture bracket comprises a main body, wherein a micro-channel is arranged in the main body, one end of the main body is provided with an inlet, the other end of the main body is provided with an outlet, and the inlet and the outlet are respectively communicated with the micro-channel; The main body comprises two groups of main branches and a bracket, the micro-channels are arranged in the bracket, two ends of the bracket are respectively connected with the inlet and the outlet through the two groups of main branches, and the micro-channels are communicated with the inlet and the outlet through the main branches; a plurality of groups of micropores are arranged on the outer wall of the bracket at intervals, and the micropores are communicated with the micro-channels; The bracket is formed by connecting a plurality of groups of branches, and a plurality of groups of mutually communicated culture holes are formed between adjacent branches; The interior of each group of the branches is provided with micro-channels extending along the branches, and the micro-channels in the groups of the branches are mutually communicated; the micropores are arranged on the outer wall of the branch at intervals, and the micropores are used for communicating the micro-channels with the culture holes. In the techni