Patent Description:
The invention provides a method for preparation of 3D brain organoids comprising:.

The traditional method of preparing human 3D brain organoids generally starts from the embryonic body, for example, preparation method of 3D brain organoids published in <NPL>et al. has made a big step forward in utilization of human pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells) in vitro to obtain 3D simulation of human brain organs. Recently, similar method published in <NPL>et al. has been applied to study the mechanism of Zika virus-induced cerebellar disease by preparing brain organoids from human pluripotent stem cells, and a small multi-well plate with stirring device has been invented to reduce the amount and cost of the factor as well as to improve the homogeneity. However, it is difficult to control the repeatability and structural similarity of the products steadily because the 3D brain organoids obtained by these two methods may contain cells and structures of other embryonic layers, and therefore applications in the field of neurological disease models and drug screening are limited, the representativeness and reliability of the obtained data are also affected.

As published in <NPL>et al. , rose-like cell clusters (mainly NPC, neural precursor cells) with the size of <NUM>,<NUM>~<NUM>,<NUM> are picked manually and induced. However, the obtained 3D new cerebral cortex has no potentials of developing into the hindbrain (negative NKX2. <NUM> staining). In addition, this method is too complicated; meanwhile, <NPL>, describes the generation and manipulation of 3D neural aggregates.

<CIT>, describes the generation of midbrain-specific organoids from human pluripotent stem cells.

<NPL>, describes self-organizing midbrain-like organoids.

simplicity, mass production as well as uniformity can't be achieved.

In view of the above, the object of the present invention is to provide a method for preparing 3D brain organoids, as defined in the claims. The 3D brain organoids with uniform size and structure can be obtained by this method, which is simple and suitable for industrialization.

The mentioned medium A comprises: retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement (Vitamin A free);.

The mentioned medium B comprises: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement (Vitamin A free).

The present invention begins with highly purified neurospheres obtained by the RONA method, guarantying that more than <NUM>% cells are neural stem cells, and thus problems existing in other methods, such as containing non-neuronal stem cells and non-brain tissue in subsequent cultures, can be well solved. According to the present invention, neurospheres are dissociated into single cells, which are plated in a fixed number after being counted, the uniformity of cell mass size and composition can be ensured, even though the size and structure of the 3D brain organoids will not be significantly different after <NUM> days' culture. Meanwhile, the medium A and the medium B used in the culture process of the present invention ensure that the cultured 3D brain organoids can be induced into the brain tissue of the forebrain, the midbrain and the hindbrain, as well as cells and structure of the six-layered brain cortex.

The present invention begins with highly purified neurospheres obtained by the RONA method, guarantying that more than <NUM>% cells are neural stem cells. The specific process of purifying neurospheres by the RONA method is referred to the article "<NPL>.

In the present invention, the neurospheres purified by the RONA method are dissociated by accutase into single cells, which are plated on a cell culture plate after being counted. Specifically, the same number of <NUM>~<NUM> cells are plated on a multi-well cell culture plate to ensure uniformity of cell cluster size after being counted. In one embodiment, an equal number of <NUM>~<NUM> cells are plated on a <NUM>-well cell culture plate with ultra-low attachment at the round bottom.

Neurospheres are cultured in medium A after being plated, and the mentioned medium A comprises: retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement (Vitamin A free). In one embodiment, the mentioned medium A comprises: <NUM>~<NUM> retinoic acid, <NUM>~<NUM> ng/mL BDNF, <NUM>~<NUM> ng/mL GDNF, <NUM>~<NUM> ascorbic acid, <NUM>~<NUM> cAMP, Neurobasal and B27 supplement (Vitamin A free). In one embodiment, the mentioned medium A comprises: <NUM> retinoic acid, <NUM> ng/mL BDNF, <NUM> ng/mL GDNF, <NUM> ascorbic acid, <NUM> cAMP, Neurobasal and B27 supplement (Vitamin A free), wherein, the dosage ratio of Neurobasal and B27 supplement (Vitamin A free) is <NUM>:<NUM>.

Cells are cultured in medium A after being plated, shaken on the low-speed orbital shaker in a humidified incubator with <NUM>% CO<NUM> at <NUM>, and then half-medium changes are performed every <NUM> to <NUM> days. It can be observed that neurospheres with uniform size are formed in each well on day <NUM>. Neurospheres are cultured in medium A until day <NUM>, and then neurospheres are transferred into medium B and cultured under the same culturing conditions.

In the present invention, the mentioned medium B comprises: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement (Vitamin A free). In one embodiment, the mentioned medium B comprises: <NUM>~<NUM> ng/mL BDNF, <NUM>~<NUM> ng/mL GDNF, <NUM>~<NUM> ascorbic acid, <NUM>~<NUM> cAMP, Neurobasal and B27 supplement (Vitamin A free). In one embodiment, the mentioned medium B comprises: <NUM> ng/mL BDNF, <NUM> ng/mL GDNF, <NUM> ascorbic acid, <NUM> cAMP, B27 supplement (Vitamin A free), wherein, the dosage ratio of Neurobasal and B27 supplement (Vitamin A free) is <NUM>:<NUM>.

Neurospheres are cultured in medium B until day <NUM>~<NUM>, and then they are encapsulated; when neurospheres are cultured in media B until day <NUM>~<NUM>, they are encapsulated for the second time and cultured continually afterwards. Homogeneous 3D organoids with simulated human brain composition can be obtained, and further encapsulation and culture can be done depending on the requirements.

Specifically, in the present invention, neurospheres are cultured until day <NUM>-<NUM>, and then they are encapsulated by Matrigel; neurospheres are encapsulated by Matrigel for the second time when they are cultured until day <NUM>~<NUM>, and cultured continually afterwards.

Specifically, in the present invention, the method for preparation further comprises the following steps: neurospheres are encapsulated for the third time when they are cultured until day <NUM>~<NUM>, and cultured continually afterwards.

Specifically, in the present invention, neurospheres are encapsulated by using Matrigel for the third time when they are cultured until day <NUM>~<NUM>, and cultured continually afterwards.

In one embodiment of the present invention, neurospheres are encapsulated when they are cultured until day <NUM>, and neurospheres are encapsulated for the second time when they are cultured until day <NUM>. Neurospheres are encapsulated for the third time when they are cultured until day <NUM>, and cultured continually afterwards.

The experimental results indicate that the present invention begins with highly purified neurospheres obtained by the RONA method, and 3D cerebral corpuscles with relatively uniform size and structure can be obtained. The 3D cerebral corpuscles can reach up to <NUM> in diameter on day <NUM> and continue to grow; meanwhile, markers such as Nestin, Tuj1, Foxg1, TBR2 and NKX2. <NUM>, etc. can be expressed in 3D cerebral corpuscles which are capable of developing into protocerebrum, deutocerebrum and tritocerebrum; in addition, 3D cerebral corpuscles can also express markers including BRN2, SATB2, CTIP2 and TBR1 with similar distribution and proportion as makers in the brain, and have the capacity to stably obtain the cerebral cortex structure.

The present invention begins with highly purified neurospheres obtained by the RONA method, and neuronal stem cells can be controlled as well as 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.

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.

The technical solutions in the embodiments of the present invention will be described clearly and completely hereinafter.

<FIG> is a flow chart of the preparation process of 3D brain organoids provided in example <NUM> of the present invention.

Step <NUM>: the neurospheres obtained by the RONA method (referring to "<NPL>) were dissociated into single cells by accutase, and then the same number of <NUM> cells were plated on a <NUM>-well cell culture plate with ultra-low attachment at the round bottom after cells were counted.

Cells were cultured in media A which was placed on the orbital shaker in a humidified incubator with <NUM>% CO<NUM> at <NUM> for <NUM> days, and half-medium changes were performed every <NUM> to <NUM> days. Medium A comprised: <NUM> retinoic acid, <NUM> ng/ml BDNF and GDNF, <NUM> ascorbic acid, <NUM> cAMP of Neurobasal and B27 supplement (Vitamin A free), wherein the dosage ratio of Neurobasal to B27 supplement was <NUM>:<NUM>.

Step <NUM>: neurospheres with uniform size could be observed in each well on day <NUM>. Medium A was replaced by media B on day <NUM>. Neurospheres were cultured continually in medium B. Medium B comprised: <NUM> ng/mL BDNF and GDNF, <NUM> ascorbic acid, <NUM> cAMP of Neurobasal and B27 supplement (Vitamin A free), wherein the dosage ratio of Neurobasal to B27 supplement was <NUM>:<NUM>.

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

Referring to <FIG>, <FIG>, <FIG> and <FIG>, <FIG> shows the image of 3D cerebral corpuscles cultured until day <NUM> provided in example <NUM> of the present invention. <FIG> is the image of 3D cerebral corpuscles cultured until day <NUM> provided in example <NUM> of the present invention. <FIG> is the image of 3D cerebral corpuscles cultured until day <NUM> provided in example <NUM> of the present invention. <FIG> is the image of 3D cerebral corpuscles cultured until day <NUM> provided in example <NUM> of the present invention. As can be seen from <FIG>, 3D cerebral corpuscles were relatively uniform in size and shape, could reach up to <NUM> in diameter on day <NUM>, 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.

Referring to <FIG> shows tissue biopsies and staining images of progenitor cells from different brain regions of 3D cerebral corpuscles cultured until week <NUM>. 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. <NUM> is a marker of hindbrain precursor cells and DAPI is a DNA dye. As shown in the <FIG>, the 3D cerebral corpuscles obtained by the method of the present invention are capable of developing into the forebrain, the midbrain and the hindbrain.

Referring to <FIG> shows tissue biopsies and staining images of neutrons from different cerebral cortex of 3D cerebral corpuscle cultured until day <NUM>. 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>, the 3D brain corpuscles obtained by the method of the present invention can express markers of the above mentioned different cortices.

Referring to <FIG> shows tissue biopsies and staining images of glial cells and neurons of 3D cerebral corpuscle cultured until day <NUM>. 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>, 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.

According to statistics, the proportion of glial cells in all cells of 3D cerebral corpuscles is about <NUM>-<NUM>%. As shown in the <FIG>, 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.

Claim 1:
A method for preparation of 3D brain organoids comprising:
dissociating neurospheres obtained by the RONA method into single cells by accutase, plating on a cell culture plate after counting the cells, and culturing in medium A for <NUM> days;
culturing the neurospheres in medium B until day <NUM>~<NUM>, and encapsulating the neurospheres; culturing the neurospheres in medium B until day <NUM>~<NUM>, followed by encapsulating said neurospheres for the second time and culturing continually afterwards;
wherein medium A comprises retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement;
wherein medium B comprises: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement; and
wherein the RONA method comprises the following steps:
a) growing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in suspension in human ESC medium without FGF2 for <NUM> days;
b)from days <NUM> to <NUM>, adding <NUM> ng/ml of noggin or <NUM> of dorsomorphin and <NUM> of SB431542 to the medium to obtain embyroid bodies (EBs);
c) on day <NUM>, transferring free-floating EBs to Matrigel- or laminin-precoated culture plates comprising N2 induction medium to allow the complete attachment of EB aggregates, and continuing the culture to allow the formation of highly compact 3D column-like neural aggregates (RONA), wherein the N2 induction medium comprises DMEM/F12, <NUM>% N2 induction supplement, <NUM> MEM non-essential amino acid solution, <NUM> GlutaMAX and <NUM>µg/ml heparin; and
d) collecting RONAs on day <NUM> and maintaining RONAs as neurospheres in suspension in Neurobasal medium containing B27 minus VitA and <NUM> GlutaMAX for <NUM> day.