Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

Summary

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.

Abstract

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.

This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.

No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Introduction

Human brain organoids are an emerging technique that allows us to recapitulate the early development of the human brain in vitro^1,2,3. Nevertheless, one of the major limitations of this model is the lack of vascularization, which plays indispensable roles not only in brain homeostasis but also in brain development⁴. In addition to the delivery of oxygen and nutrients, accumulating evidence suggests that the vascular system of the brain regulates neural differentiation, migration, and synaptogenesis during development^5,6. Therefore, there is an urgent need to establish reliable models that can provide the missing vascular signaling and structure to brain organoids, enhancing the complexity of human brain organoid generation⁷.

Among the proposed methods for vascularization, two main streamlines can be considered: organoid engrafting into a living organism and purely in vitro technologies co-culturing endothelial cells and neural cells^8,9,10,11,12. Intracerebral transplantation in mice is costly and time-consuming, making other technologies relevant for simpler models. The chick chorioallantoic membrane (CAM) assay has been used extensively to study angiogenesis^13,14,15. In the last decade, several groups have successfully engrafted different types of organoids, including kidney^16,17, cardiac¹⁸, and tumor organoids^19,20, into CAMs. Nevertheless, little is known about the efficacy, toxicity/rejection, physiological effect, and methods to engraft human brain organoids into the CAM. Another interesting and yet unexplored aspect is the formation of a chimeric blood-brain barrier (BBB) between the CAM and the organoid astrocytic interface. Previous pioneering work suggested the putative feasibility of generating a BBB in the CAM by transplanting astrocytes and astrocyte-conditioned medium^21,22,23. However, mature astrocytes seem to be unable to achieve this^24,25. Thus, the astrocyte-induced formation of the BBB remains debatable, and transplanting human brain organoids would allow us to shed light on this controversy.

This video article describes a protocol for an in ovo human brain organoid transplant into CAM that promotes growth, improvement, and vascularization, resulting in organoids that encompass histologically compatible BBB elements. Here, we present a protocol ensuring the survival of the chicken embryo and report on the permissivity of the CAM to sustain brain organoid growth.

Protocol

The White Leghorn chicken (Gallus gallus) embryos were treated by following the Guide for the Care and Use of Laboratory Animals from the Institute of Laboratory Animals Resources, Commission of Life Sciences, National Research Council, USA, and the experiments were approved by the Council for Care and Use of Experimental Animals from the University of Barcelona.

1. Non-guided brain organoid preparation

1. Maintain H9 human embryonic stem cells (hESCs) in mTESR1 prewarmed at room temperature (RT) under a confluence of 70% in 6-well plates coated with Matrigel dissolved in DMEM (1:40) in a 5% CO₂ incubator at 37 °C.
   NOTE: Matrigel must be kept in ice until its use as a coating to prevent its polymerization.
2. Generate brain organoids following the unguided brain protocol ².
   1. Dissociate H9 hESCs using 500 µL of the Cell Dissociation Solution and incubate for 3-5 min at 37 °C. Resuspend in mTESR1 containing a ROCK inhibitor Y27632 (final concentration 50 µM) and seed 9,000 cells per well in a low adhesion pretreated 96-well plate.
   2. Once the organoids aggregate in mTESR1 containing a ROCK inhibitor Y27632 (final concentration 50 µM), transfer them into induction medium for 5-6 days.
   3. Following unguided brain organoid protocol²⁶, transfer the organoids to neural induction media at ~days 4-5.
   4. Once they acquire the size and the neuroepithelium ring, transfer them into an ice-cold Matrigel drop. Once the Matrigel is polymerized, transfer the organoids to a 5 cm, low-adhesion Petri dish with 5 mL of prewarmed maturation medium.
   5. After 4 days, put the brain organoids under orbital agitation.
3. Collect the organoids for harvesting in fresh DPBS immediately before grafting.

2. Egg maintenance, development activation, and eggshell puncture

1. Store fertilized eggs (at day 0) in an incubator at 18 °C until use.
   NOTE: At this temperature, they do not develop, remaining at the same stage of maturation.
2. When required for use, incubate them at 38 °C and 60% relative humidity with soft rocking rotation at an angle of 45 °C.
3. On day 1, clean the eggshell surface with 70% ethanol and dry it with a paper towel to sterilize it.
4. Place a surgical paper adhesive bandage, hereafter referred to as "bandage," on top of the egg, covering the air chamber of the egg.
   NOTE: This will prevent dust from the eggshell from falling into the CAM where it gets stuck.
5. Perform the air chamber perforation puncture, hereafter referred to as air chamber hole (ACH), with a sterile needle 21 G x 11/2'', softly puncturing the eggshell. Open the ACH on the upper tip of the egg to avoid disturbing the CAM, which at this point lays separated from the shell.
6. To find the best position to puncture the ACH, use a lamp to determine the thinnest eggshell surface at the upper tip of the egg. The direct light beam transmission indicates the best position to drill.
7. Cover the ACH with a new bandage to protect the embryo from the external environment and place the egg back into the incubator.
8. From this moment on, cease rotation in the incubator and maintain the eggs in a vertical position with the window on top.
   NOTE: This will facilitate the development of the embryo, and the CAM becomes available at the top with a chamber of air between the embryo and the eggshell, allowing for subsequent grafting.
9. Open the ACH from day 1 to day 4. Viability increases on day 4 (3 days prior to grafting day).

3. Brain organoid grafting

1. Perform grafting on day 7 when the CAM is well developed and vascularized, covering the egg all over the embryo.
2. Remove the bandage, and wipe the eggshell surface with 70% ethanol to sterilize the eggshell, prior to carefully widening the ACH into a grafting window.
3. Place a new, bigger bandage covering the extension beyond the final grafting window to avoid fragments of eggshell falling into the CAM while enlarging the grafting window.
   NOTE: This bigger window is needed to select the best grafting position and for subsequent grafting.
4. Enlarge the grafting window by softly puncturing the eggshell until the window reaches a diameter of approximately 2 cm. Softly remove the edges of the eggshell window with tweezers until the window is enlarged. Remove the remaining dust and eggshell pieces that will remain attached to the bandage by softly peeling off the bandage.
5. Place the organoid with a P200 automatic pipet. Cut the tips with sterile and sharp scissors to enlarge the opening accordingly to the size of the organoid and prevent damage to the organoid or use wide-gauge transferring sterile tips. Collect the organoid in a maximum volume of 50 µL of PBS and transfer it directly into the CAM, on a side opposite to the location of the embryo.
6. Ensure that the position of the graft is distant from the yolk to avoid its rupture during harvesting, and preferably, place it near a blood vessel.
7. Place a drop of 7 µL of Matrigel surrounding the organoid after placing the organoid onto the CAM.
   NOTE: This will prevent the organoid from moving around the membrane.
8. Close the window using a small piece of parafilm fitting the exact size of the window and attach it to the shell with an adhesive bandage.
   NOTE: It is important to fully cover the window with the parafilm to avoid the embryo from drying out while allowing gas exchange. Delimit properly the extension of the window coverage, avoiding exceeding the window, as it could hinder the necessary gas exchange for correct embryo development²⁷.

4. Transplanted organoid harvesting

1. Harvest the grafted organoids and the surrounding CAM on day 12 of chicken development, corresponding to 38 Hamburger and Hamilton stage of development (HH38)²⁸, 5 days after grafting. At this stage, the CAM is fully differentiated with mature vasculature while the feather germs coverts the wings as well as the upper eyelid of the embryo.
2. Upon removing the adhesive bandage, further enlarge the window to facilitate sample harvest using scissors and fine tweezers.
3. Visualize the organoid with the help of a binocular dissection microscopy if needed, followed by a 2 min prefixation step with 4% paraformaldehyde (PFA) to help organoid and CAM harvesting.
4. Collect a 1.5 cm² section of the CAM containing the organoid.
5. Sample fixation
   1. Wash the samples with 1x PBS and fix them with 4% PFA (in PBS, pH 7.2) for 30 min at 4 °C. Then, wash the samples for 3 x 5 min in PBS.
   2. Store the samples at 4 °C in PBS with sodium azide or directly cryoprotect them in 30% sucrose-PBS overnight at 4 °C.
   3. Upon cryoprotection, embed the samples in optimal cutting temperature compound (OCT) and store them at -20 °C until sectioning. Cut 20 µm thick tissue slices using a cryostat and collect them on xylanized slides. Store the slides at -20 °C until they are used for staining.

5. Immunofluorescence

1. Remove OCT. from the sections with a PBS solution with 0.1% Triton X-100 (PBS-T) for 10 min at room temperature (RT).
2. Treat the sections with a blocking solution (BS) with a 2% BSA, 3% donkey serum PBS-T 0.01% solution for 1 h at RT.
3. Dilute the selected primary antibody (see the Table of Materials) in BS and incubate samples overnight at 4 °C.
4. The next day, wash the samples 3 x 10 min in 1x PBS at RT.
5. Dilute the secondary antibody in BS and incubate for 2 h at RT (see the Table of Materials).
6. Incubate the samples for 10 min with 4',6-diamidino-2-phenylindole (DAPI) to mark the cell nuclei at RT, diluted in PBS-T at a 1:1,000 dilution.
7. Mount the slides with mounting medium and cover with glass coverslips.
8. Take all images using a widefield fluorescence microscope at 2.5, 10, 20, and 40x.
9. Store the slices at 4 °C.

6. Hematoxylin and eosin (H&E) staining

NOTE: To prove that the grafting worked, perform H&E staining.

1. Remove OCT with PBS-T 0.1% for 10 min at RT.
2. Perform serial dehydration in a hood as follows: 10 min in distilled water (DW), 45 s in hematoxylin, 1 min in DW, 20 s in PBS, 2 x 30 s in 70% EtOH, 2 x 30 s in 80% EtOH, 2 x 30 s in 96% EtOH, 30 s in eosin, 2 x 15 s in 96% EtOH, 2 x 15 s in 100% EtOH, 1 min in xylol-100% ETOH, 1 min in xylol, 1 min in xylol (fresh).
3. Mount the glass coverslips with a xylene-based mounting medium.

Results

Selecting the embryo maturation schedule for the transplant
The experiment begins at D0 when fertilized eggs are incubated at 38 °C and 60% relative humidity. The chorioallantoic membrane (CAM) is a highly vascularized extraembryonic membrane that develops after egg incubation. It is formed by the fusion of the allantois and chorion. At D1, after 24 h of incubation, the air chamber is punctured to prevent the CAM from attaching to the inner shell membrane. Puncturing the air chamber at D1 impr...

Discussion

In this study, we describe a detailed protocol with numerous key steps that provide favorable growth and development of human brain organoids upon grafting without perturbing the survival of the chicken embryos. We recommended the use of sterile needles to puncture the air chamber of the egg after 24 h of incubation (day 1). Additionally, we also tried to make the puncture at day 4 (after checking through the eggshell by light to test the development of the vasculature to be sure that we were working only with healthy em...

Materials

Name	Company	Catalog Number	Comments
Anti-TUBB3 [Tuj1], mouse	BioLegend	801201	1:1,000
Anti-GFAP, rabbit	GeneTex	GTX108711	1:500
Anti-rabbit AlexaFluor 488, goat.	Invitrogen	A-21206	1:1,000
Anti-mouse AlexaFluor 594, goat	Jackson ImmunoResearch	715-585-150	1:500
Fertilized White Leghorn chicken (Gallus gallus) eggs	Granja Gibert (Cambrils, Spain)
DAPI	Invitrogen	D1306	1:10,000
DPX	Sigma	100579	xylene-based mounting medium
Gentle Dissociation Solution	CreativeBiolabs	ITS-0622-YT187	cell dissociation solution
Matrigel	BD Biosciences	356234
Mowiol 4-88 mounting media	Merk	81381
Paper towel, lab-grade	Sigma-Aldrich	Z188956
ROCK inhibitor Y27632	Millipore	SCM075	10 nM
Sharp-Point Surgical Scissors	VWR	470106-340
Superfrost Plus Adhesion Microscope Slides	Epredia	J1800AMNZ