人口腔粘膜类器官培养基

I. Pre-experimental preparation :
1 、 15 mL After disinfecting the surfaces of sterile centrifuge tubes, pipettes, pipettes, sterile gun tips, etc., they are placed in an ultra-clean workbench for ultraviolet irradiation. 30 min 。
2 , in advance 30 min From 4℃ Remove the oral mucosal organoid culture medium and equilibrate it to room temperature beforehand. 1h From -20℃ The desired volume of extracellular matrix gel was removed from the freezer and melted on ice.

II. Preparation of Human oral mucosa cell suspension
1 Remove tissue cleansing fluid and tissue digestive enzymes from the tissue in advance. -20℃ Remove from the refrigerator and place in 4 ℃ Melt.
2 Sterilize the surface of the ice box and then move it into the ultra-clean table. Under aseptic conditions, place petri dishes on ice boxes and place tissue samples in tissue wash solution for washing. 3-5 s 。
3 Under aseptic conditions, the washed tissue sample is immersed in a container... 1-3mL In a Petri dish containing tissue cleansing solution. The sample was mechanically cut into pieces on ice using a sterile scalpel. 1-2 mm³ After sizing, transfer the sample fragments to 15 mL In sterile centrifuge tubes.
4 Join 5-10 mL The tissue cleaning liquid is placed in a centrifuge tube, 1500 rpm 4℃ centrifuge 5 min 。
5 Abandon the Shangqing and join 3-5mL Tissue digestive enzymes (the volume of digestive enzymes is at least the volume of tissue) 5 (times), mark the sample name and number, seal it with sealing film, place it in a constant temperature oscillating incubator for digestion, and set the parameters. 37 ℃ 、 200-250 rpm (Set rotation speed according to sample texture), digestion 20 min Digestion can be terminated when the tissue is observed to appear flocculent.
6 Take out the digested sample and add... 5 mL Contains 10% serum DMEM Complete culture medium was used to stop digestion. 100 μm Cell sieve filtration.
7 Suspend the sieved cells in 1500 rpm room temperature centrifugation 5 min 。
8 After abandoning the Shangqing Dynasty, he DMEM Complete medium was resuspended and counted to obtain a separated cell suspension for use.

3. Oral mucosal organoids cultivate
1 The obtained cell suspension 1500 rpm Room temperature centrifugation 5 min After discarding the supernatant, it was pre-cooled on ice, and the volume of matrix gel added was calculated to ensure that the final concentration of oral mucosal organoids was... 5×10⁶ microscopic cell / milliliters.
2 Using a precooled gun tip, add the calculated volume of matrix gel to the cell pellet and gently mix well (the entire process is performed on ice to avoid bubbles). Then, mix the mixture of cells and matrix gel according to... 30 μl/ The holes are hemispherical in shape and dispensed with glue. 24 Aperture plate.
3 Invert the culture plate 37 ℃ 、 5 % CO₂ At least in the conditioned incubator 30 min 。
4 After the matrix glue has completely solidified, along 24 Add slowly to the hole wall 1mL/ The wells are restored to the oral mucosa organoid medium at room temperature in advance, and the culture plate is placed in an incubator, 37 ℃ 、 5 % CO₂ Stand and culture under these conditions.
5 Observe the formation of organoids under a microscope, each 3~4 The culture medium is changed once a day.

IV. Oral mucosal organoid collection
1 Discard the culture medium and add to each well. 1 mL Precooled serum-free DMEM/F12 The culture medium is then used to moisten the gun tip, and the gun tip is repeatedly blown until the dome-shaped gel droplets completely detach from the bottom of the plate. The organoid mixture after being blown into the wells is collected. 15 mL In the centrifuge tube, 1500 rpm Room temperature centrifugation 5 min Gently suck off the supernatant and let it settle for later use.
2 Add to the precipitate 3-5mLTrypLE^TM Express enzyme (Gibco#12605010) ， 37℃ ， 200 rpm Dissociation on horizontal shaker 10 min Left and right (the specific dissociation time depends on the size and number of organoids, based on a significant reduction of half of the visible particles. The dissociated organoids can be observed under a microscope halfway through, and the organoids will dissociate to an average concentration of 5~10 The optimal cell mass size is [number of cells].
3 After the dissociation is complete, add an equal volume 10% Fetal bovine serum DMEM Digestion of the culture medium was terminated at room temperature. 1500 rpm Centrifugation 5min He abandoned the Shangqing Dynasty and waited for use.

V. Oral mucosal organoid passage
1 The collected cell pellets were resuspended and counted using oral mucosal organoid culture medium.
2 Re-inoculate organoids according to the organoid culture steps and continue to expand the culture.

Oral mucosal organoid culture medium is a medium that promotes the formation and expansion of human oral mucosal organoids in vitro. This product is a sterile liquid mixture system containing amino acids, vitamins, organic and inorganic compounds, and growth factors essential for maintaining the growth of the target cells. The formulation of the culture medium can provide a suitable nutritional environment to selectively promote the growth and amplification of human oral mucosa organoids in vitro.