Brain organoid cryopreservation solution and brain organoid cryopreservation and revival method

Abstract

The present invention relates to a brain organoid freezing solution and a method for freezing and reviving brain organoids. The components of the brain organoid freezing solution are 5-10% DMSO, 5-10μM Y27632 and additives, and the balance is cell culture fluid, and the additives are selected from one or more of 0.1-5% methylcellulose, 0.1-20% ethylene glycol, and 0.1-20% polyvinyl pyrrolidone. At the same time, including pretreatment with 10μM Y27632 before freezing, 10μM Y27632 static culture treatment technology after recovery, and organoid embedding technology, normal freezing and recovery of brain organoids are achieved and complete physiological activity is preserved. The present invention greatly saves brain organoid culture time and preparation cost, is conducive to the homogenization, large-scale culture, preparation and storage of brain organoids, and will greatly promote the development of basic research in brain science and clinical transformation applications.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a brain organoid cryopreservation solution and a brain organoid cryopreservation and revival method. Background Art

[0002] In recent years, organoid technology is one of the most important advances in the field of stem cell research. With the vigorous development of organoid technology, brain organoids have shown great potential in studying human brain development, establishing disease models and neural regeneration (Madeline A et al., Nature, 2013)(Abed A et al., 2018, Nat Biotechnol). However, brain organoid culture has difficulties such as long culture cycle, high cost and lack of freezing technology, which greatly limits the transformation and application of brain organoids. Therefore, the development of brain organoid freezing technology is of great significance for the application transformation of organoids, the development of neural regeneration, and the screening of clinical drugs.

[0003] Brain organoids are neural tissues finely constructed by a variety of neural cells, containing neural functions and certain neural network activities. At present, the cryopreservation of brain organoids faces problems such as morphological collapse, disintegration, neuronal apoptosis, and destruction of neural network structure after cryopreservation and thawing. The current organ cryopreservation technology can only realize the cryopreservation of ordinary embryonic stem cells, gametes, embryos (Leibo Set al., 2019, Cryobiology) (Lu Zhang et al., 2016, Sci Rep), glioma organoids (Fadi Jacob et al., 2020, Nat Protoc), endometrial organoids (Riley E et al., 2021, Cryobiology), etc. In 1951, the earliest bovine sperm could be successfully frozen to -79°C in 10% glycerol, fertilized in vitro after thawing, and successfully gave birth to calves. 21 years later, bovine embryos could be directly frozen in 2.0M DMSO and frozen to -196°C at a rate of 0.2°C / min, and then thawed at a rate of 360°C / min. However, the birth rate is still limited. Glioma organoids and endometrial organoids have strong proliferation and regeneration capabilities, and can amplify the surviving cells after cryopreservation and resuscitation by passaging, so they can break through the damage caused by the cryopreservation process and complete the structural remodeling process. Brain organoids contain neurons without mitotic ability and neural tissue with specific cell spatial structure, which do not have the ability of passaging and self-renewal. Therefore, the cryopreservation methods of 2D cells and non-neural organoids cannot meet the cryopreservation needs of brain organoids. Summary of the invention

[0004] Based on the current situation that the 2D cell and non-neural organoid cryopreservation methods in the prior art cannot meet the cryopreservation needs of brain organoids, the present invention provides a brain organoid cryopreservation solution and a method for cryopreservation and revival of brain organoids.

[0005] The present invention develops the best cryopreservation solution formula for brain organoids and develops a method for cryopreservation and resuscitation of brain organoids, thereby maintaining the normal morphology of brain organoids after cryopreservation and resuscitation, neuron survival, and integrity of neural tissue structure and network activity. It is expected to reduce the cycle and cost of brain organoid culture and better apply them to basic research, clinical application and drug screening.

[0006] Specifically, the present invention develops and designs cryopreservation components based on the requirements of maintaining the morphology of brain organoids after cryopreservation and resuscitation, neuronal apoptosis, and the integrity of neural tissue structure and network activity, based on the basic principles of cell and tissue cryopreservation and the functional characteristics of various organic and inorganic substances, and integrating existing cryopreservation technologies. At the same time, key technical steps such as pretreatment culture before cryopreservation, cryopreservation fluid pretreatment, and post-recovery pretreatment culture are improved, ultimately achieving non-destructive cryopreservation and resuscitation of brain organoids.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides a brain organoid cryopreservation solution, which comprises 5-10% DMSO, 5-10 μM Y27632 and additives, and the balance is a cell culture solution, wherein the additive is selected from one or more of 0.1-5% methylcellulose, 0.1-20% ethylene glycol, and 0.1-20% polyvinyl pyrrolidone; in the cryopreservation solution of the present invention, the concentrations of all substances are final concentrations, that is, compared with the percentage of the final cryopreservation solution, the concentration of the solid component is mass / volume percentage, and the concentration of the liquid component is volume percentage;

[0009] The brain organoid freezing solution is suitable for different types of brain organoids, including cortical organoids, ventral telencephalic organoids, spinal cord organoids, etc.

[0010] In one embodiment of the present invention, the brain organoid freezing solution comprises 10% DMSO, 10 μM Y27632 and additives, and the remainder is cell culture medium. The additive is selected from one or more of 1% methyl cellulose, 10% ethylene glycol, and 10% polyvinyl pyrrolidone.

[0011] In one embodiment of the present invention, the brain organoid freezing solution comprises 10% DMSO, 10 μM Y27632, 1% methyl cellulose and 10% ethylene glycol, with the remainder being cell culture medium.

[0012] In the description of the culture medium in the following embodiments of the present invention, the concentrations of all substances are final concentrations, that is, the concentration of solid components is mass / volume percentage, and the concentration of liquid components is volume percentage compared to the percentage of the final culture medium.

[0013] The present invention also provides a method for freezing brain organoids, comprising the following steps:

[0014] Cryopreserved forebrain organoids were cultured normally in brain organoid culture medium containing 5-10 μM Y27632 (preferably 10 μM) for 0.5-24 h (preferably 1.5 h).

[0015] Transfer the brain organoids to the freezing solution and place them at room temperature for pretreatment for 1 / 6-5h (preferably 0.5h-1h);

[0016] Cryopreserving the brain organoids in the cryopreservation solution;

[0017] The brain organoid is selected from one or more of cortical organoids, ventral telencephalic organoids, and spinal cord organoids.

[0018] In one embodiment of the present invention, the brain organoid culture medium is selected as Neurobasal maturation medium, the components of which are 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, and 200 μM Vc.

[0019] In the present invention, the purpose of normally culturing the frozen forebrain organoids in a brain organoid culture medium containing 5-10 μM (preferably 10 μM) Y27632 is to enhance the survival ability and growth state of the organoids.

[0020] In the present invention, the purpose of transferring the brain organoids to the cryopreservation solution and pre-treating them at room temperature for 1 / 6-5 hours is to allow the protective permeable components in the cryopreservation solution to fully enter the organoid, play a protective role on the entire organoid, and give full play to the protective effect of the cryopreservation solution on various cells in the organoid (such as reducing vitrification, increasing osmotic pressure, etc.). At the same time, the time of room temperature pre-treatment needs to be appropriately adjusted according to the size of the organoid.

[0021] In the present invention, the brain organoids are transferred to the freezing solution and pretreated at room temperature for 0.5h-1h, which is mainly for brain organoids with an average diameter of about 3mm. It should be noted that the room temperature pretreatment time should be appropriately extended as the size of the organoids increases and maintained at 0.5h / 2mm.

[0022] In one embodiment of the present invention, the method for cryopreserving the brain organoids in the cryopreservation solution is a conventional cryopreservation method. The present invention provides a specific operation method: placing the cryopreservation solution containing the brain organoids in a gradient cooling cryopreservation box, placing it in a -80°C environment, and transferring it to a liquid nitrogen tank or a temperature environment close to the liquid nitrogen tank for long-term storage overnight or after 8-12 hours.

[0023] The present invention also provides a method for resuscitating a brain organoid, comprising the following steps:

[0024] S1. After taking out the frozen brain organoids from liquid nitrogen, thaw them quickly at 25-37°C within no more than 5 minutes.

[0025] S2, the rapidly thawed brain organoids were cultured in a medium containing 10 μM Y27632 for 24 h;

[0026] S3, then replace with fresh normal medium without Y27632 for cultivation, change the fresh medium every day for the first three days, and then change the medium every other day;

[0027] S4. During the culture process in step S3, Matrigel embedding treatment is performed on the 4th day to allow Matrigel to evenly wrap the revived brain organoids, and then normal culture is continued, and the medium is changed every other day;

[0028] The brain organoid is selected from one or more of cortical organoids, ventral telencephalic organoids, and spinal cord organoids.

[0029] In one embodiment of the present invention, the culture medium in the recovery process is selected to be suitable for culturing brain organoids;

[0030] Preferably, the composition of the culture medium used for resuscitation of organoids in the first week of cryopreservation is: SRM + 100nM LDN193189 + 10μM SB431542 (depending on the type of induced organoid, the additives will also change accordingly); the composition of the culture medium used for resuscitation of organoids in the second to third weeks of cryopreservation is: Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax by volume + 1% double antibody by volume + 20ng / mL EGF + 20ng / mL bFGF;

[0031] The composition of the culture medium used for reviving organoids at the 4th week of cryopreservation and older is: Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax by volume + 1% double antibody by volume + 10 ng / mL BDNF + 10 ng / mL GDNF + 200 μM Vc.

[0032] In one embodiment of the present invention, in step S2, the conditions for static culture are: static culture in an incubator at 37° C. and 5% CO 2 .

[0033] The purpose of culturing the rapidly thawed brain organoids in a culture medium containing 10 μM Y27632 for 24 hours in the present invention is to allow the newly revived organoids to quickly recover their viability under the action of Y27632, and to avoid the situation where the organoid structure is destroyed due to the external force generated by the rotation of the culture gene when the organoids are directly placed on a shaker.

[0034] In one embodiment of the present invention, in step S3, the culture conditions are: 37° C., continuous culture on a horizontal shaker with a rotation speed of 10 to 100 rpm (eg, 90 rpm).

[0035] In one embodiment of the present invention, in step S4, the method for Matrigel embedding treatment is: thawing Matrigel on ice, then tilting the culture plate, carefully aspirating and discarding the old culture medium, carefully aspirating an appropriate amount of Matrigel (determined by the size of the organoid, and ultimately needed to cover the entire organoid) with a pre-cooled gun tip and evenly dropping it on the organoid, keeping the tilt angle in the incubator and tilting it for 10-30 minutes, then slowly adding culture medium, gently picking up the Matrigel-wrapped brain organoids from the bottom of the culture dish and suspending them in the culture medium, and placing them on a shaker for normal culture.

[0036] In one embodiment of the present invention, in step S1, the cryopreserved brain organoid refers to a brain organoid cryopreserved using the brain organoid cryopreservation solution of the present invention.

[0037] In one embodiment of the present invention, in step S1, the cryopreserved brain organoid refers to a brain organoid cryopreserved using the brain organoid cryopreservation solution of the present invention and the cryopreservation method provided by the present invention.

[0038] The present invention provides a new brain organoid cryopreservation technology based on optimal cryopreservation components, improved cryopreservation methods and resuscitation methods.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] The present invention study found that the optimal formula combination of the new brain organoid freezing solution is: 10% DMSO + 10μMY27632 + 1% methyl cellulose + 10% ethylene glycol.

[0041] At the same time, the present invention found that pretreatment with 10 μM Y27632 before cryopreservation, static culture treatment technology with 10 μM Y27632 after recovery, and organoid embedding treatment can achieve normal cryopreservation and recovery of brain organoids and preserve complete physiological activity.

[0042] The present invention greatly saves the culture time and preparation cost of brain organoids, is conducive to the realization of uniformity, large-scale culture, preparation and storage of brain organoids, and will greatly promote the development of basic research in brain science and clinical translational applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The morphology of brain organoids after 48 hours of cryopreservation and recovery with different cryopreservation solutions, including Figure 1 A, Figure 1 B, Figure 1 C, Figure 1 D, Figure 1 E, Figure 1 F, Figure 1 G. All scale bars are 2 mm.

[0044] Figure 1 A: 10% DMSO, 1% methylcellulose, and 10 μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The morphology of the frozen brain organoids 48 hours after recovery is intact with a small amount of cell debris.

[0045] Figure 1 B: 10% DMSO, 10% ethylene glycol, and 10 μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The morphology of the frozen brain organoids 48 hours after recovery was intact with a small amount of cell debris.

[0046] Figure 1 C: 10% DMSO, 10% polyvinyl pyrrolidone (PVP), and 10μMY27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The morphology of the frozen brain organoids 48 hours after recovery is intact with a small amount of cell fragments.

[0047] Figure 1 D: 10% DMSO, 1.0 M trehalose, and 10 μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The morphology of the frozen brain organoids 48 hours after recovery is intact with a small amount of cell debris.

[0048] Figure 1 E: 10% DMSO, 0.3M glucose (Glucose), and 10μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The morphology of the frozen brain organoids 48 hours after recovery is intact with a small amount of cell fragments.

[0049] Figure 1F: 10% DMSO, 0.3M sucrose (Sucrose), and 10μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The morphology of the frozen brain organoids 48 hours after recovery showed incomplete morphology, disintegration of the organoids, and large pieces of cell fragments.

[0050] Figure 1 G: 10% DMSO, 2.0M proline, and 10μM Y27632 were added to the culture medium of the brain organoids in the fourth week to prepare the freezing solution. The morphology of the frozen brain organoids after thawing for 48 hours showed a large amount of cell debris. The dotted line represents the scattered cell debris.

[0051] Figure 2 Axon growth chart of brain organoids after cryopreservation and resuscitation with different cryopreservation solutions on day 14, including Figure 2 A, Figure 2 B, Figure 2 C, Figure 2 D, Figure 2 E, Figure 2 F, Figure 2 G, Figure 2 H, Figure 2 I, Figure 2 J, Figure 2 K, Figure 2 L. All scale bars are 100 μm.

[0052] Figure 2 A: Axon growth status of normally cultured brain organoids.

[0053] Figure 2 B: Brain organoids were cryopreserved and revived in combination 1: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% ethylene glycol cryopreservation solution until the 14th day of axon growth, with a large number of long and dense axons extending outward.

[0054] Figure 2 C: Brain organoids were cryopreserved and revived in combination 2: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone cryopreservation solution until the 14th day of axon growth, with long and dense axons extending outward.

[0055] Figure 2 D: Brain organoids were cryopreserved and revived in combination 3: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% ethylene glycol + 10% polyvinyl pyrrolidone cryopreservation solution and cultured until the 14th day in the axon growth state, with axons extending outward.

[0056] Figure 2E: Brain organoids were cryopreserved and revived in combination 4: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone + 0.3M glucose cryopreservation solution and cultured until the 14th day in the axon growth state, with a few long axons extending outward.

[0057] Figure 2 F: 10% DMSO, 1% methylcellulose, and 10 μM Y27632 were added to the culture medium of brain organoids at week 4 to prepare the freezing solution. The organoids were cryopreserved and revived until the 14th day, when axons grew, with long and dense axons extending outward.

[0058] Figure 2 G: 10% DMSO, 10% ethylene glycol, and 10 μM Y27632 were added to the culture medium of brain organoids at week 4 to prepare a freezing solution. The organoids were cryopreserved and revived until the 14th day when axons grew densely, connecting into a network.

[0059] Figure 2 H: 10% DMSO, 10% polyvinyl pyrrolidone (PVP), and 10μM Y27632 were added to the culture medium of brain organoids at week 4 to prepare the freezing solution. The organoids were cryopreserved and resuscitated to the axon growth state on day 14, with short and dense axon growth.

[0060] Figure 2 I: 10% DMSO, 1.0M trehalose, and 10μM Y27632 were added to the culture medium of brain organoids at week 4 to prepare a freezing solution. The organoids were cryopreserved and resuscitated until the axon growth state on day 14, with almost no living cells present.

[0061] Figure 2 J: 10% DMSO, 0.3M sucrose (Sucrose), and 10μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The organoids were cryopreserved and revived until the axon growth state on the 14th day. A few living cells existed, but there was no axon growth.

[0062] Figure 2 K: 10% DMSO, 0.3M glucose (Glucose), and 10μM Y27632 were added to the culture medium of brain organoids in the 4th week to prepare the freezing solution. The organoids were cryopreserved and revived until the axon growth state on the 14th day, and no axon growth was observed.

[0063] Figure 2L: 10% DMSO, 2.0 M proline, and 10 μM Y27632 were added to the culture medium of brain organoids at week 4 to prepare a freezing solution. The organoids were cryopreserved and revived until the axon growth state on day 14, and no axon growth was observed.

[0064] Figure 3 Immunofluorescence staining of MAP2 / Sox2 on day 14 of cryopreservation and resuscitation of brain organoids using different cryopreservation solutions, including Figure 3 A, Figure 3 B, Figure 3 C, Figure 3 D, Figure 3 E, Figure 3 F, Figure 3 G, Figure 3 H, Figure 3 I, Figure 3 J, Figure 3 K, Figure 3 L. All scale bars are 500 μm.

[0065] Figure 3 A: Normally cultured brain organoids.

[0066] Figure 3 B: Brain organoids were cryopreserved and revived on day 14 in combination 1: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol cryopreservation solution.

[0067] Figure 3 C: Brain organoids cryopreserved and revived on day 14 in combination 2: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone cryopreservation solution

[0068] Figure 3 D: Brain organoids were cryopreserved and revived on day 14 in combination 3: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol + 10% polyvinyl pyrrolidone cryopreservation solution.

[0069] Figure 3 E: Brain organoids were cryopreserved and revived on the 14th day in combination 4: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone + 0.3 M glucose cryopreservation solution.

[0070] Figure 3 F: In the 4th week, 10% DMSO, 1% methylcellulose, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0071] Figure 3G: In the 4th week, 10% DMSO, 10% ethylene glycol, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0072] Figure 3 H: In the 4th week, 10% DMSO, 10% polyvinyl pyrrolidone (PVP), and 10 μM Y27632 were added to the culture medium to prepare the cryopreservation solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0073] Figure 3 I: In the 4th week, 10% DMSO, 1.0 M trehalose, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0074] Figure 3 J: In the 4th week, 10% DMSO, 0.3M sucrose, and 10 μM Y27632 were added to the culture medium to prepare the cryopreservation solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0075] Figure 3 K: In the 4th week, 10% DMSO, 0.3 M glucose, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0076] Figure 3 L: In the 4th week, 10% DMSO, 2.0 M proline, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0077] Figure 4 Caspase3 / Tuj-1 immunofluorescence staining of brain organoids after cryopreservation and resuscitation with different cryopreservation solutions on day 14, including Figure 4 A, Figure 4 B, Figure 4 C, Figure 4 D, Figure 4 E, Figure 4 F, Figure 4 G, Figure 4 H, Figure 4 I, Figure 4 J, Figure 4 K, Figure 4 L. All scale bars are 500 μm.

[0078] Figure 4 A: Normally cultured brain organoids.

[0079] Figure 4B: Brain organoids were cryopreserved and revived on day 14 in combination 1: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol cryopreservation solution.

[0080] Figure 4 C: Brain organoids cryopreserved and revived on day 14 in combination 2: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone cryopreservation solution

[0081] Figure 4 D: Brain organoids were cryopreserved and revived on day 14 in combination 3: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol + 10% polyvinyl pyrrolidone cryopreservation solution.

[0082] Figure 4 E: Brain organoids were cryopreserved and revived on the 14th day in combination 4: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone + 0.3 M glucose cryopreservation solution.

[0083] Figure 4 F: In the 4th week, 10% DMSO, 1% methylcellulose, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0084] Figure 4 G: In the 4th week, 10% DMSO, 10% ethylene glycol, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0085] Figure 4 H: In the 4th week, 10% DMSO, 10% polyvinyl pyrrolidone (PVP), and 10 μM Y27632 were added to the culture medium to prepare the cryopreservation solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0086] Figure 4 I: In the 4th week, 10% DMSO, 1.0 M trehalose, and 10 μM Y27632 were added to the culture medium to prepare the cryopreservation solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0087] Figure 4 J: In the 4th week, 10% DMSO, 0.3 M sucrose, and 10 μM Y27632 were added to the culture medium to prepare the cryopreservation solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0088] Figure 4K: In the 4th week, 10% DMSO, 0.3 M glucose, and 10 μM Y27632 were added to the culture medium to prepare the cryopreservation solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0089] Figure 4 L: In the 4th week, 10% DMSO, 2.0 M proline, and 10 μM Y27632 were added to the culture medium to prepare the freezing solution, and the brain organoids were cryopreserved and revived until the 14th day.

[0090] Figure 5 The morphology of brain organoids after 48 hours of cryopreservation and recovery using different combinations of cryopreservation solutions, including Figure 5 A, Figure 5 B, Figure 5 C, Figure 5 D. All scale bars are 2 mm.

[0091] Figure 5 A: The morphology of brain organoids 48 hours after cryo-recovery in the combination 1: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% ethylene glycol cryopreservation solution. The morphology is intact with almost no cell debris.

[0092] Figure 5 B: Morphology of brain organoids 48 hours after cryo-recovery in combination 2: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone cryopreservation solution. The morphology is intact with almost no cell debris.

[0093] Figure 5 C: Morphology of brain organoids 48 hours after cryostasis in combination 3: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% ethylene glycol + 10% polyvinyl pyrrolidone cryopreservation solution. The morphology is intact with almost no cell debris.

[0094] Figure 5 D: Morphology of brain organoids 48 hours after cryopreservation and recovery in combination 4: 10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% polyvinyl pyrrolidone + 0.3M glucose cryopreservation solution. The morphology is intact with almost no cell debris. DETAILED DESCRIPTION

[0095] The present invention first provides a brain organoid cryopreservation solution, which comprises 5-10% DMSO, 5-10 μM Y27632 and additives, and the balance is a cell culture solution, wherein the additive is selected from one or more of 0.1-5% methylcellulose, 0.1-20% ethylene glycol, and 0.1-20% polyvinyl pyrrolidone; in the cryopreservation solution of the present invention, the concentrations of all substances are final concentrations, that is, compared with the percentage of the final cryopreservation solution, the concentration of the solid component is mass / volume percentage, and the concentration of the liquid component is volume percentage;

[0096] The brain organoid freezing solution is suitable for different types of brain organoids, including cortical organoids, ventral telencephalic organoids, spinal cord organoids, etc.

[0097] In one embodiment of the present invention, the brain organoid freezing solution comprises 10% DMSO, 10 μM Y27632 and additives, and the remainder is cell culture medium. The additive is selected from one or more of 1% methyl cellulose, 10% ethylene glycol, and 10% polyvinyl pyrrolidone.

[0098] In one embodiment of the present invention, the brain organoid freezing solution comprises 10% DMSO, 10 μM Y27632, 1% methyl cellulose and 10% ethylene glycol, with the remainder being cell culture medium.

[0099] In the description of the culture medium in the following embodiments of the present invention, the concentrations of all substances are final concentrations, that is, the concentration of solid components is mass / volume percentage, and the concentration of liquid components is volume percentage compared to the percentage of the final culture medium.

[0100] The present invention also provides a method for freezing brain organoids, comprising the following steps:

[0101] Cryopreserved forebrain organoids were cultured normally in brain organoid culture medium containing 5-10 μM Y27632 for 0.5-24 h.

[0102] Transfer the brain organoids to the freezing medium and pretreat at room temperature for 1 / 6-5h;

[0103] Cryopreservation of brain organoids in cryopreservation medium.

[0104] In one embodiment of the present invention, the brain organoid culture medium is selected as Neurobasal maturation medium, the components of which are 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, and 200 μM Vc.

[0105] In the present invention, the brain organoids are transferred to the freezing solution and pretreated at room temperature for 0.5h-1h, which is mainly for brain organoids with an average diameter of about 3mm. It should be noted that the room temperature pretreatment time should be appropriately extended as the size of the organoids increases.

[0106] In one embodiment of the present invention, the method for cryopreserving the brain organoids in the cryopreservation solution is a conventional cryopreservation method. The present invention provides a specific operation method: placing the cryopreservation solution containing the brain organoids in a gradient cooling cryopreservation box, placing it in a -80°C environment, and transferring it to a liquid nitrogen tank or a temperature environment close to the liquid nitrogen tank for long-term storage overnight or after 8-12 hours.

[0107] The present invention also provides a method for resuscitating a brain organoid, comprising the following steps:

[0108] S1. After taking out the frozen brain organoids from liquid nitrogen, thaw them quickly at about 37°C (25-37°C) within no more than 5 minutes;

[0109] S2, the rapidly thawed brain organoids were cultured in a medium containing 10 μM Y27632 for 24 h;

[0110] S3, then replace with fresh normal medium without Y27632 for cultivation, change the fresh medium every day for the first three days, and then change the medium every other day;

[0111] S4. During the culture process in step S3, Matrigel embedding treatment is performed on the 4th day to allow Matrigel to evenly wrap the revived brain organoids, and then normal culture is continued, and the medium is changed every other day.

[0112] In one embodiment of the present invention, the culture medium in the recovery process is selected to be suitable for culturing brain organoids;

[0113] Preferably, the composition of the culture medium used for resuscitation of organoids in the first week of cryopreservation is: SRM + 100nM LDN193189 + 10μM SB431542 (depending on the type of induced organoid, the additives will also change accordingly); the composition of the culture medium used for resuscitation of organoids in the second to third weeks of cryopreservation is: Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax by volume + 1% double antibody by volume + 20ng / mL EGF + 20ng / mL bFGF;

[0114] The composition of the culture medium used for reviving organoids at the 4th week of cryopreservation and older is: Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax by volume + 1% double antibody by volume + 10 ng / mL BDNF + 10 ng / mL GDNF + 200 μM Vc.

[0115] In one embodiment of the present invention, in step S2, the conditions for static culture are: static culture in an incubator at 37° C. and 5% CO 2 .

[0116] In one embodiment of the present invention, in step S3, the culture conditions are: 37° C., and continuous culture on a horizontal shaker with a rotation speed of 80 to 100 rpm (eg, 90 rpm).

[0117] In one embodiment of the present invention, in step S4, the method for Matrigel embedding treatment is: thawing Matrigel on ice, then tilting the culture plate, carefully aspirating and discarding the old culture medium, carefully aspirating an appropriate amount of Matrigel (determined by the size of the organoid, and ultimately needed to cover the entire organoid) with a pre-cooled gun tip and evenly dropping it on the organoid, keeping the tilt angle in the incubator and tilting it for 10-30 minutes, then slowly adding culture medium, gently picking up the Matrigel-wrapped brain organoids from the bottom of the culture dish and suspending them in the culture medium, and placing them on a shaker for normal culture.

[0118] In one embodiment of the present invention, in step S1, the cryopreserved brain organoid refers to a brain organoid cryopreserved using the brain organoid cryopreservation solution of the present invention.

[0119] In one embodiment of the present invention, in step S1, the cryopreserved brain organoid refers to a brain organoid cryopreserved using the brain organoid cryopreservation solution of the present invention and the cryopreservation method provided by the present invention.

[0120] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0121] Example 1

[0122] Preliminary determination of candidate components for brain organoid cryopreservation solution

[0123] 1. Preliminary screening steps for brain organoid cryopreservation solution formulations

[0124] a: Embryonic stem cells H9 were induced to initially form brain organoid spheres under the action of SRM medium (100nM LDN193189, 10μM SB431542), and then replaced with Neurobasal induction medium (Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax by volume + 1% double antibody by volume + 20ng / mL EGF + 20ng / mL bFGF) in the second week until the end of the third week. After the fourth week, Neurobasal maturation medium (Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax + 1% double antibody by volume + 10ng / mL BDNF + 10ng / mL GDNF + 200μM Vc) was used to form brain organoids at different developmental stages.

[0125] b: Next, select 1% methylcellulose, 10% ethylene glycol, 10% polyvinyl pyrrolidone (PVP), 1.0M trehalose, 2.0M proline, 0.3M glucose, and 0.3M sucrose as additives for ordinary embryonic stem cell freezing solution (culture medium + 10% DMSO), and then add 10μM Y27632 and mix evenly.

[0126] c: Before freezing, add Y27632 at a final concentration of 10 μM to the normal cultured organoid culture medium, continue culturing for 1.5 hours, then carefully transfer the brain organoids to the freezing solution, leave at room temperature for 30 minutes, then place in a gradient cooling freezing box, place in a -80℃ refrigerator, and transfer to a liquid nitrogen tank for long-term storage overnight.

[0127] d: Take the frozen organoids out of liquid nitrogen, thaw them quickly at 37°C (no more than 5 minutes), and then carefully transfer them to pre-warmed culture medium (any culture medium suitable for brain organoid culture. These include: the culture medium composition used for resuscitation of organoids in the first week of cryopreservation is: SRM+100nM LDN193189+10μM SB431542 (additives will also change accordingly depending on the type of induced organoid); the culture medium composition used for resuscitation of organoids in the second to third weeks of cryopreservation is: Neurobasal+2%B27+1%NEAA+1%GlutaMax+1%Double Antibody+20ng / mL EGF+20ng / mL bFGF; the culture medium composition used for resuscitation of organoids in the fourth week and older weeks of cryopreservation is: Neurobasal+2%B27+1%NEAA+1%GlutaMax+1%Double Antibody+10ng / mL BDNF+10ng / mL GDNF+200μM Vc. And this method is applicable to different types of brain organoids, including: cortical organoids, GABA organoids, spinal cord organoids, etc. ), placed in a 37°C, 5% CO2 incubator for 24 hours, the medium was changed every day for the first three days, and then changed every other day (Note: Y27632 with a final concentration of 10μM was added to the culture medium on the first day, and fresh normal culture medium without Y27632 was replaced on the second day, and the culture was continued on a horizontal shaker with a rotation speed of about 90rpm).

[0128] e: Observation 48 hours after resuscitation, methylcellulose group ( Figure 1 A), ethylene glycol group ( Figure 1 B), polyvinyl pyrrolidone group ( Figure 1 C), trehalose group ( Figure 1 D), glucose group ( Figure 1 E), the amount of cell debris is relatively small, and the proline group has a large amount of cell debris ( Figure 1 G), brain organoids in the sucrose group disintegrated ( Figure 1 F).

[0129] f: On the 4th day after recovery, the organoids were embedded with Matrigel (thaw Matrigel on ice, then tilt the culture plate, carefully aspirate the old culture medium, use a pre-cooled pipette tip to carefully aspirate an appropriate amount of Matrigel (depending on the size of the organoid, which should eventually cover the entire organoid) and evenly drop it on the organoid, keep the tilt angle and place it in the incubator for 10-30 minutes, then slowly add culture medium, gently lift the Matrigel-wrapped brain organoids from the bottom of the culture dish and suspend them in the culture medium, and place them on a shaker for normal culture.) Allow Matrigel to evenly wrap the revived brain organoids, and continue normal culture, changing the medium every other day. On the 8th day after recovery, which was the 4th day after embedding, the brain organoids in the methylcellulose group, ethylene glycol group, and polyvinyl pyrrolidone group began to show accelerated metabolism, and the culture medium turned yellow. On the 14th day after recovery, the methylcellulose group ( Figure 2 F), ethylene glycol group ( Figure 2 G) Brain organoid outgrowth and unfrozen organoids ( Figure 2 A) The axons of similar length, the axons of the polyvinyl pyrrolidone group are relatively shorter ( Figure 2 H), trehalose group ( Figure 2 I), sucrose group ( Figure 2 J), glucose group ( Figure 2 K), no obvious axon extension was observed in the proline group ( Figure 2 L).

[0130] g: The brain organoids on the 14th day after thawing were fixed, dehydrated, frozen and sectioned as usual, and immunofluorescence staining was used to identify the survival and structural integrity of the frozen thawing organoids. The results of Sox2 / MAP2 immunofluorescence staining showed that: the methylcellulose group ( Figure 3 F), ethylene glycol group ( Figure 3 G), polyvinyl pyrrolidone group ( Figure 3 H) About 2 / 3 of the structure of the organoids remained intact, and the trehalose group ( Figure 3 I), sucrose group ( Figure 3 J), glucose group ( Figure 3 K), proline group ( Figure 3 L) There are almost no Sox2 / MAP2 positive cells in the organoids. Further Tuj1 / Caspase3 immunofluorescence staining results showed that: methylcellulose group ( Figure 4 F), ethylene glycol group ( Figure 4 G), polyvinyl pyrrolidone group ( Figure 4 H) The surviving Tuj1+ cells in the organoids had normal morphology, and only a small number of cells underwent apoptosis, while the trehalose group ( Figure 4 I), sucrose group ( Figure 4 J), glucose group ( Figure 4 K), proline group ( Figure 4 L) A large number of cells in the organoid undergo apoptosis.

[0131] Therefore, based on the above research, the candidate components of brain organoid freezing solution were preliminarily determined to be: methylcellulose, ethylene glycol, and polyvinyl pyrrolidone.

[0132] Example 2

[0133] Determination of the formula combination of brain organoid cryopreservation solution

[0134] According to the method in Example 1, the precise formula of brain organoid cryopreservation solution was further explored. Four combinations were set up, namely:

[0135] Combination 1: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol;

[0136] Combination 2: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% polyvinylpyrrolidone;

[0137] Combination 3: 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol + 10% polyvinylpyrrolidone;

[0138] Combination 4: 10% DMSO+10 μM Y27632+1% methylcellulose+10% polyvinylpyrrolidone+0.3 M glucose.

[0139] The experimental results showed that there was almost no cell debris after thawing of organoids frozen using combination 1-4 ( Figure 5 ); Normal axon growth can be observed, and the axon growth effect of brain organoids after cryopreservation and thawing in combination 1 is the best ( Figure 2 BE); by immunofluorescence staining, the organoids frozen in combination 1-4 showed less apoptosis after thawing than those frozen in the single additive group, and the structure of nerve cells and neurites were more intact. Among the several combinations, the brain organoids frozen and thawed in combination 1 were closer to the morphology and neural network of brain organoids in normal development ( Figure 3 BE, Figure 4 BE). Therefore, the combination of 10% DMSO + 10 μM Y27632 + 1% methylcellulose + 10% ethylene glycol was determined to be the optimal cryopreservation formula combination for brain organoids.

[0140] Example 3

[0141] Optimization of methods for cryopreservation and thawing of brain organoids

[0142] The present invention provides an improvement in the pre-cryopreservation technology of brain organoids: before cryopreservation, the brain organoids need to be normally cultured in a 10 μM Y27632 culture medium for 1.5 hours, then transferred to a cryopreservation solution, and pretreated at room temperature for 0.5 hours to 1 hour (for brain organoids with an average diameter of about 3 mm, that is, the room temperature pretreatment time should be appropriately extended as the size of the organoids increases), and then a conventional cryopreservation process is performed.

[0143] The present invention provides a technical improvement in the brain organoid resuscitation process: the rapidly thawed brain organoids need to be statically cultured in 10μM Y27632 culture medium for 24 hours, and then replaced with fresh normal culture medium without Y27632 on the next day and cultured on a horizontal shaker with a rotation speed of about 90 rpm / min. The fresh culture medium is replaced every day for the first three days, and the medium is replaced every other day thereafter. Matrigel embedding treatment is performed on the 4th day.

[0144] In summary, the present invention has discovered a new cryopreservation solution formula combination for brain organoids (10% DMSO + 10μM Y27632 + 1% methylcellulose + 10% ethylene glycol), and simultaneously adopted 10μM Y27632 pretreatment before cryopreservation and 10μM Y27632 static culture treatment technology and organoid embedding technology after recovery, ultimately achieving normal cryopreservation and recovery of brain organoids. The present invention greatly saves brain organoid culture time and preparation costs, is conducive to the homogenization, large-scale culture, preparation and storage of brain organoids, and will greatly promote the development of basic research in brain science and clinical transformation applications.

[0145] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of brain organoids, characterized in that: The following steps are involved: Cryopreserved forebrain organoids were cultured normally in brain organoid culture medium containing 5-10 μM Y27632 for 0.5-24 h. Transfer the brain organoids to the brain organoid freezing solution and pretreat at room temperature for 1 / 6-5h; Cryopreserving the brain organoids in the cryopreservation solution; The brain organoid is selected from one or more of cortical organoids, ventral telencephalic organoids, and spinal cord organoids; The brain organoid freezing solution comprises 10% DMSO, 10 μM Y27632 and additives, and the remainder is cell culture medium, wherein the additive is selected from one or more of 1% methylcellulose, 10% ethylene glycol, and 10% polyvinyl pyrrolidone; The brain organoid is selected from one or more of cortical organoids, ventral telencephalic organoids, and spinal cord organoids.

2. The method for cryopreservation of brain organoids according to claim 1, characterized in that: The brain organoid freezing solution comprises 10% DMSO, 10 μM Y27632, 1% methyl cellulose and 10% ethylene glycol, and the remainder is cell culture medium.

3. The method for cryopreservation of brain organoids according to claim 1, characterized in that: The brain organoid culture medium is selected as Neurobasal maturation medium, the components of which are 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, and 200 μM Vc.

4. The method for cryopreservation of brain organoids according to claim 1, characterized in that: The method for freezing brain organoids in a cryopreservation solution is as follows: placing the cryopreservation solution containing the brain organoids in a gradient cooling cryopreservation box, placing it in a -80°C environment, and transferring it to a liquid nitrogen tank or a temperature environment close to the liquid nitrogen tank for long-term storage overnight or after 8-12 hours.

5. A method for resuscitating a brain organoid, characterized in that: The following steps are involved: S1. After taking out the frozen brain organoids from liquid nitrogen, thaw them quickly at 25-37°C within no more than 5 minutes. S2, the rapidly thawed brain organoids were cultured in a medium containing 10 μM Y27632 for 24 h; S3, then replace with fresh normal medium without Y27632 for cultivation, change the fresh medium every day for the first three days, and then change the medium every other day; S4. During the culture process in step S3, Matrigel embedding treatment is performed on the 4th day to allow Matrigel to evenly wrap the revived brain organoids, and then normal culture is continued, and the medium is changed every other day; The brain organoid is selected from one or more of cortical organoids, ventral telencephalic organoids, and spinal cord organoids; In step S1, the cryopreserved brain organoid refers to a brain organoid cryopreserved by the method according to any one of claims 1 to 4.

6. The method for resuscitation of brain organoids according to claim 5, characterized in that: The culture medium for the recovery process should be suitable for brain organoid culture; The composition of the culture medium used for organoid recovery in the first week of cryopreservation is: SRM+100nM LDN193189+10μM SB431542; the composition of the culture medium used for organoid recovery in the second to third weeks of cryopreservation is: Neurobasal+2% B27+1% NEAA+1% GlutaMax+1% double antibody+20ng / mL EGF+20ng / mL bFGF; The composition of the culture medium used for reviving organoids at the 4th week of cryopreservation and older is: Neurobasal + 2% B27 by volume + 1% NEAA by volume + 1% GlutaMax by volume + 1% double antibody by volume + 10 ng / mL BDNF + 10 ng / mL GDNF + 200 μM Vc.

7. The method for resuscitation of brain organoids according to claim 5, characterized in that: In step S2, the conditions for static culture are: static culture in an incubator at 37° C. and 5% CO 2 .

8. The method for resuscitation of brain organoids according to claim 5, characterized in that: In step S3, the culture conditions are: 37° C., and continuous culture on a horizontal shaker with a rotation speed of 10 to 100 rpm.

9. The method for resuscitation of brain organoids according to claim 5, characterized in that: In step S4, the method for Matrigel embedding treatment is: thaw Matrigel on ice, then tilt the culture plate, discard the old culture medium, use a pre-cooled pipette tip to suck up Matrigel and evenly drop it on the organoid, and finally the Matrigel needs to cover the entire organoid, keep the tilted angle and place it in the incubator for 10-30 minutes, then add culture medium, pick up the Matrigel-wrapped brain organoids from the bottom of the culture dish and suspend them in the culture medium, and place them on a shaker for normal culture.

Images