Human tube organ culture medium kit Plus

Human Airway Organoid Kit Plus

Human tube organ culture medium kitPlus

1 Product Description

Model based Biovascular Organ Culture Kit（Human Airway Organoid Kit Plus）It is a chemically defined cell culture medium used to establish and maintain human airway organs derived from human airway stem cells. The self-renewal of airway epithelium is driven by the proliferation of basal stem cells. The human respiratory tract organs include basal cells, ciliated cells, secretory cells, and a small number of neuroendocrine cells. Therefore, the organs exhibit all the characteristics of the airway epithelium in terms of system structure, cell type composition, and self-renewal dynamics. Therefore, they have great potential for airway development and disease research.

2 Product Information

3 Other self provided materials and reagents

4 Instructions for using human respiratory organ culture medium

1 After receiving the organoid culture medium, place the medium in4℃Thaw the refrigerator;

2 After thawing the culture medium, invert it upside down and mix thoroughly. Then, pack it according to daily needs in a biosafety cabinet or clean workbench. It is recommended to pack it into10mL/Management;

3 Please seal and store the packaged culture medium in-20℃Take out the packaged culture medium and let it equilibrate at room temperature before use.

5 Establishment and Passage Culture of Human Vascular Organs

Attention: Research involving primary human tissue materials must comply with all relevant institutional and government regulations. Prior to collecting primary human tissue materials, informed consent must be obtained from all subjects.

a) Establishment of primary human respiratory tract organs

1 Primary tissue samples should be in vitro5minPlace pre cooled living tissue preservation solution inside（2-8℃）In the sample tube, the sample needs to be covered with tissue preservation solution (if the sample has already been placed in other buffer solutions or culture media, please replace the entire solution with pre cooled tissue preservation solution). Low temperature（2~8℃）Transport and transfer to the laboratory.

2 Place the matrix adhesive before the experiment4℃Thaw on ice while removing the culture medium and allowing it to equilibrate at room temperature. Centrifugal tubes, test tubes, or plastic tips in contact with cells need to be rinsed with organoid culture anti adhesion moisturizing solution before use.

3 In a clean workbench, transfer the sample to a culture dish and evaluate whether the obtained tissue is composed of epithelium. If there is fat or muscle tissue present, use a knife or forceps under an dissecting microscope to remove as much of these non epithelial components as possible. If there is no fat or muscle tissue, please proceed to the next step immediately.

4 Use epithelial organ basic culture medium or double antibody containing mediumDPBSRinse the tissue twice.

5 Use tweezers to transfer tissue to1.5mLIn a centrifuge tube, crush it on ice using sterile methods0.5~2mm²size.

6 Use the shredded tissue50Resuspend twice the volume of the tissue and transfer to15mLBlow and strike the resuspended tissue block inside the centrifuge tube. Yu37℃，100rpmHorizontal oscillation digestion in a constant temperature shaker under certain conditions15-30Minutes, every10Observe the digestion of the tissue for minutes. When the tissue fragments are clearly dispersed and the suspension is turbid, take an appropriate amount of tissue digestion suspension for microscopic examination. Stop digestion when there are more viable cell clusters in the suspension. If the tissue fragments are large or digestion is not possible, extend the digestion time appropriately. During digestion, different specifications can be used (in order from large to small)10mLThe5mLThe1mL）The pipette is used to blow the tissue digestion suspension, helping to fully digest it. When most tissue fragments can pass through1mLWhen the pipette tip is used, the digestion process is completed.

7 Translate into EnglishFBSAdd to the tissue digestion mixture, with a final concentration of2%And use it100μmCell filter filtration.

8 Collect the filtrate and4℃Below is250gcentrifugation3minute In the presence of visible red sediment, discard the supernatant and add2mLResuspend and precipitate red blood cell lysate, and lyse red blood cells at room temperature1Minutes, and within4℃Below is250gcentrifugation3minute

9 Discard the supernatant and resuspend the precipitate in epithelial organoid basal culture medium4℃Below is250gcentrifugation3Minute, repeat this step again to remove digestive fluid andFBSTake an appropriate amount of suspension for cell viability testing before centrifugation.

10 Discard the supernatant and add an appropriate amount of cell pellet to the matrix gel according to the cultivation requirements（>70%）And mix well on ice (note: the mixing and blowing action should be gentle, avoiding the generation of a large number of bubbles, and mixing at room temperature needs to be controlled within15Within seconds, mix well and place on ice. Note: The matrix adhesive should be stored on ice to prevent it from solidifying. Carry out the process as soon as possible. approximately10,000Cells should be seeded in25μLIn the matrix adhesive. Do not excessively dilute the matrix adhesive (the proportion of matrix adhesive should be>70%(Volume of matrix adhesive)/(Total volume), as this may inhibit the correct formation of solid droplets.

11 Place the mixed suspension of matrix gel and cells into the bottom of the culture well plate, and use a pipette tip to slightly level the suspension, taking care to avoid contact with the side wall of the well plate. (Recommended:96Plate inoculation3~10μL/Hole,48Plate inoculation10~20μL/Hole,24Plate inoculation20~30μL/Hole).

Attention: Once the organoid is resuspended in the matrix gel, proceed with seeding as soon as possible, as the matrix gel may solidify in the test tube or pipette tip.

12 Place the culture plate in37℃and5%CO₂In the incubator15-25Minute, let the matrix gel solidify.

13 After the matrix gel solidifies, slowly add room temperature equilibrated organoid culture medium along the wall to avoid damaging the already solidified structure. (Recommended:96Add orifice plate100μL/Hole,48Add orifice plate250μL/Hole,24Add orifice plate500μL/Hole). Note: Do not add the culture medium directly to the top of the matrix gel droplets, as this may damage the already solidified structure.

14 Place the culture plate in37℃and5%CO₂In a constant temperature incubator.

15 every2~3Change the culture medium once a day, carefully extract the culture medium from the well, and add fresh room temperature balanced organoid culture medium.

16 Closely monitor the growth status of organoids, ideally, human respiratory organoids should7-14Built within the day.

b) Passage culture of human respiratory organs

1 Cultivate the organoid to a diameter of100~500μmWhen the size is small (or when it turns black and no longer grows), the passage of organoids can be carried out (approximately per generation)1~2week The following consumables that come into contact with cells must be rinsed with a moisturizing solution before use:

2 Discard the old culture medium, add an equal volume of epithelial organoid basic culture medium, gently scrape (or blow) off the matrix gel and organoid mixture with a cell scraper or pipette tip, and transfer to1.5mLIn centrifuge tubes (maximum collection per tube)2~3Pore like organs, avoid excessive volume and low digestion efficiency), blow and beat5~10Next, separate the organoids from the matrix gel,300gcentrifugation3minute

3 Remove the supernatant and add5Dissolve organoid digestive fluid equal to the volume of the matrix gel and organoid mixture, blow and mix well, and then place it in37℃Digestion in the incubator5~10minute Take out, blow and mix well10μLMicroscopic examination of the mixed solution to determine if it has been digested into small cell clusters. If digestion is insufficient, the digestion time can be appropriately extended. If cell counting is required, it needs to be digested into individual cells, and the digestion time can be extended to10~20Terminate digestion in minutes and perform cell counting using pan blue staining in the background. Closely monitoring the digestion process minimizes the incubation time in organoid dissociation fluid.

4 After digestion is complete, add5Blow and mix the basic culture medium of the double volume epithelial organ to terminate the digestion reaction, and then250gcentrifugation3minute

5 Discard the supernatant and wash with epithelial organ basic culture medium2Next, remove residual digestive fluids. Secondary cleaning can merge and collect organoids in the same centrifuge tube

6 After cleaning, the cells in the centrifugal precipitate are passaged and cultured, and matrix gel is added to the cell precipitate（>70%）And mix well on ice (note that the mixing and blowing action should be gentle, avoiding the generation of a large number of bubbles, and mixing at room temperature needs to be controlled within15Within seconds, mix well and place on ice. Attention: The dilution ratio of the matrix adhesive should be within70%The above is to ensure the structural stability of the matrix gel during the cultivation process.

7 Place the mixed suspension of matrix gel and cells into the bottom of the culture well plate, and use a pipette tip to slightly level the suspension, taking care to avoid contact with the side wall of the well plate. (Recommended:96Plate inoculation3~10μL/Hole,48Plate inoculation10~20μL/Hole,24Plate inoculation20~30μL/Hole). Attention: To prevent the matrix adhesive from solidifying at room temperature, this step should be completed as soon as possible.

8 Place the culture plate in37℃and5%CO₂In the incubator15-25Minute, let the matrix gel solidify.

9 After the matrix gel solidifies, slowly add room temperature equilibrated organoid culture medium along the wall to avoid damaging the already solidified structure. (Recommended:96Add orifice plate100μL/Hole,48Add orifice plate250μL/Hole,24Add orifice plate500μL/Hole). Note: Do not add the culture medium directly to the top of the matrix gel droplets, as this may damage the already solidified structure.

10 Place the culture plate in37℃and5%CO₂In a constant temperature incubator.

11 every2~3Change the culture medium once a day, carefully extract the culture medium from the well, and add fresh room temperature balanced organoid culture medium.

12 Closely monitor the growth status of organoids until they require further experimentation

V2.1edition

Update time:2025/5/27