Method for preparing 3D brain organoids

Abstract

The present invention provides a method for preparing 3D brain organoids, comprising the following steps: neurospheres obtained by the RONA method are dissociated into single cells by accutase, plated on a cell culture plate after being counted, cultured in medium A until day 7; neurospheres are cultured in medium B until day 2535, and then they are encapsulated by Matrigel; neurospheres are further cultured in media B until day 5565, and then they are encapsulated by Matrigel for the second time and cultured continually afterwards. The present invention also provides a medium for culturing 3D brain organoids. The present invention begins with highly purified neurospheres obtained by the RONA method, and neuronal stem cells can be controlled and cultured to achieve true 3D brain organoids with uniform size and structure by this relatively simple method. The 3D brain organoids have six-layered cortical structure of the brain and various subtypes of inhibitory interneuron cells, which are suitable for disease research in vitro, drug screening, etc., and are of great significance in industrialization.

Claims

1. A method for preparation of 3D brain organoids comprising: (a) deriving a first batch of neurospheres from rosette neural aggregates (RONAs); (b) dissociating the first batch of neurospheres into single cells; (c) counting the single cells; (d) plated the single cells on a cell culture plate; (e) culturing the plated single cells in medium A for 7 days to obtain a second batch of neurospheres; (f) culturing the second batch of neurospheres in medium B until day 25-35; (g) encapsulating the second batch of neurospheres in a solubilized basement membrane preparation; (h) further culturing the encapsulated second batch of neurospheres in medium B until day 55-65; and (i) encapsulating the further cultured encapsulated second batch of neurospheres for a second time in the solubilized basement membrane preparation; and (j) continuously culturing the encapsulated second batch of neurospheres following the second encapsulation in the solubilized basement membrane preparation; wherein the medium A comprises: retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium, and a neuronal cell culture supplement; and wherein the medium B comprises: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium, and the neuronal cell culture supplement.

2. The method for preparation according to claim 1, wherein substantially the same number of the single cells are plated on each well in a multi-well cell culture plate; and wherein the number of the single cells plated on each well ranges from 1000 to 50000.

3. The method for preparation according to claim 1, wherein the medium A comprises: 1-5 M retinoic acid, 10-30 ng/mL BDNF, 10-30 ng/mL GDNF, 0.1-0.5 mM ascorbic acid, 5-15 M cAMP, Neurobasal medium, and the neuronal cell culture supplement; wherein the medium B comprises: 10-30 ng/mL BDNF, 10-30 ng/mL GDNF, 0.1-0.5 mM ascorbic acid, 5-15 M cAMP, Neurobasal medium, and the neuronal cell culture supplement; and wherein the neuronal cell culture supplement is Vitamin A-free.

4. The method for preparation according to claim 3, wherein the medium A comprises: 2 M retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 10 M cAMP, Neurobasal medium, and the neuronal cell culture supplement; wherein the medium B comprises: 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 10 M cAMP, Neurobasal medium, and the neuronal cell culture supplement; and wherein the neuronal cell culture supplement is Vitamin A-free.

5. The method for preparation according to claim 1, comprising: (k) further culturing the encapsulated second batch of neurospheres following the second encapsulation in medium B until day 85-100; (l) encapsulating the further cultured encapsulated second batch of neurospheres for a third time in the solubilized basement membrane preparation; and (m) continuously culturing the second batch of neurospheres following the third encapsulation in the solubilized basement membrane preparation.

6. The method for preparation according to claim 5, wherein in step (f), the second batch of neurospheres are cultured until day 30; in step (h), the encapsulated second batch of neurospheres are cultured until day 60; and in step (k), the encapsulated second batch of neurospheres following the second encapsulation in the solubilized basement membrane preparation are cultured until day 90.

Description

DESCRIPTION OF THE DRAWINGS

(1) In order to illustrate embodiments of the present invention or technical solutions of the existing technology more clearly, the drawings used in the description of embodiments or the existing technology will be briefly introduced. Obviously, the drawings in the following description only relates to the embodiments of the present invention. Other appended drawings can also be obtained by ordinary technicians in the field from the provided drawings of the present invention without making any creative efforts.

(2) FIG. 1 is the flow chart of the preparation process of 3D brain organoids provided in example 1 of the present invention;

(3) FIG. 2 is the image of 3D cerebral corpuscles cultured until day 17 provided in example 1 of the present invention;

(4) FIG. 3 is the image of 3D cerebral corpuscles cultured until day 50 provided in example 1 of the present invention;

(5) FIG. 4 is the image of 3D cerebral corpuscles cultured until day 50 provided in example 1 of the present invention;

(6) FIG. 5 is the image of 3D cerebral corpuscles cultured until day 88 provided in example 1 of the present invention;

(7) FIG. 6 shows tissue biopsies and staining images of progenitor cells from different brain regions of 3D cerebral corpuscles cultured until week 10;

(8) FIG. 7 shows tissue biopsies and staining images of neutrons from different cerebral cortex of 3D cerebral corpuscles cultured until day 88;

(9) FIG. 8 shows tissue biopsies and staining images of glial cells and neurons of 3D cerebral corpuscles cultured until day 63;

(10) FIG. 9 shows tissue biopsies and staining image of glial cells and neurons of 3D cerebral corpuscles cultured until day 63.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

(11) The technical solutions in the embodiments of the present invention will be described clearly and completely hereinafter. Obviously, only part of the embodiments of the present invention is involved herein. All other embodiments acquired by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

Example 1

(12) FIG. 1 is a flow chart of the preparation process of 3D brain organoids provided in example 1 of the present invention.

(13) Step 1: the neurospheres obtained by the RONA method (referring to Cultured Networks of Excitatory Projection Neurons and Inhibitory Interneurons for Studying Human Cortical Neurotoxicity published in Science Translational Medicine by Xu J C and Fan J in April, 2016) were dissociated into single cells by accutase, and then the same number of 5000 cells were plated on a 96-well cell culture plate with ultra-low attachment at the round bottom after cells were counted.

(14) Cells were cultured in media A which was placed on the orbital shaker in a humidified incubator with 5% CO.sub.2 at 37 C. for 7 days, and half-medium changes were performed every 3 to 5 days. Medium A comprised: 2 M retinoic acid, 20 ng/ml BDNF and GDNF, 0.2 mM ascorbic acid, 10 M cAMP of Neurobasal and B-27 supplement (Vitamin A free), wherein the dosage ratio of Neurobasal to B-27 supplement was 50:1.

(15) Step 2: neurospheres with uniform size could be observed in each well on day 2. Medium A was replaced by media B on day 7. Neurospheres were cultured continually in medium B. Medium B comprised: 20 ng/mL BDNF and GDNF, 0.2 mM ascorbic acid, 10 M cAMP of Neurobasal and B-27 supplement (Vitamin A free), wherein the dosage ratio of Neurobasal to B-27 supplement was 50:1.

(16) Step 3: neurospheres were cultured in Media B until day 30, and then they were encapsulated by MATRIGEL on the surface of non-hydrophilic sterile materials. Neurospheres were cultured on a 96-well culture plate until day 60, and then they were encapsulated by MATRIGEL for the second time. Neurospheres were cultured in media B until day 90, homogeneous 3D organoids with simulated human brain composition could be obtained, and further encapsulation and culture can be made depending on the requirements.

(17) Referring to FIG. 2, FIG. 3, FIG. 4 and FIG. 5, FIG. 2 shows the image of 3D cerebral corpuscles cultured until day 17 provided in example 1 of the present invention. FIG. 3 is the image of 3D cerebral corpuscles cultured until day 50 provided in example 1 of the present invention. FIG. 4 is the image of 3D cerebral corpuscles cultured until day 50 provided in example 1 of the present invention. FIG. 5 is the image of 3D cerebral corpuscles cultured until day 88 provided in example 1 of the present invention. As can be seen from FIG. 2 to FIG. 5, 3D cerebral corpuscles were relatively uniform in size and shape, could reach up to 4 mm in diameter on day 88, and continue to grow. However, the 3D cerebral corpuscles obtained by most of other methods are hard to grow up to such a size and remain healthy within the same time duration.

(18) Referring to FIG. 6, FIG. 6 shows tissue biopsies and staining images of progenitor cells from different brain regions of 3D cerebral corpuscles cultured until week 10. Among them, Nestin is a common marker protein expressed by neural precursor cells. Tuj1 is a protein marker commonly expressed by nerve cells. Foxg1 is a marker of forebrain precursor cells. TBR2 is a marker of mid-brain subventricalzone and neural precursor cells of hippocampus. NKX2.1 is a marker of hindbrain precursor cells and DAPI is a DNA dye. As shown in the FIG. 6, the 3D cerebral corpuscles obtained by the method of the present invention are capable of developing into the forebrain, the midbrain and the hindbrain.

(19) Referring to FIG. 7, FIG. 7 shows tissue biopsies and staining images of neutrons from different cerebral cortex of 3D cerebral corpuscle cultured until day 88. Among them, REELIN, BRN2, SATB2, CTIP2, and TBR1 are markers of neurons from the cerebral cortex I/II, III, IV, V, and VI, respectively. As can be seen from FIG. 7, the 3D brain corpuscles obtained by the method of the present invention can express markers of the above mentioned different cortices.

(20) Referring to FIG. 8, FIG. 8 shows tissue biopsies and staining images of glial cells and neurons of 3D cerebral corpuscle cultured until day 63. Among them, nNOS, PV and SST are markers of inhibitory brain neurons. MAP2 is a marker of relatively mature nerve cells, and DAPI is a DNA dye. As shown in the FIG. 8, the 3D cerebral corpuscles obtained by the method of the present invention at least contain such three inhibitory brain neurons, which play an important role in brain development and function, and the 3D cerebral corpuscles are relatively mature.

(21) According to statistics, the proportion of glial cells in all cells of 3D cerebral corpuscles is about 50-70%. As shown in the FIG. 9, 3D cerebral corpuscles obtained by the method described in the present invention contain glial cells which play an important role in brain development and function, and their proportion and distribution are very close to those of the human brain.

(22) The above described examples are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in the technical field, improvements and embellishments can be made without departing from the scope of the principles of the present invention, and these improvements and embellishments shall also be regarded as the scope of protection of the present invention.