Wet Lab Protocols
DNA sourcing
The templates and sources for the various PCR products are listed below:
|
PCR Product Used in Gibson |
Template Source Plasmid |
Source Company/Notes |
|
pcDNA5 backbone |
pcDNA5-FRT-mCh |
Addgene, #127109 |
|
hUBC promoter |
FUGW-hUBC |
Gersbach Lab, Duke |
|
CMV promoter |
pcDNA5-FRT-mCh |
Addgene, #127019 |
|
cLuc gene |
pIND-cLuc |
Addgene, #53224 |
|
tdTomato gene |
pCRISPR_HOT-tdTomato |
Addgene, #138567 |
|
DhdR-NLS-FLAG gene, FLAG-NLS-DhdR gene |
Directly synthesized gene block |
Twist Biosciences |
PCR primers were purchased from IDT and the sequences used are shown here. The DNA constructs used for the binding sites were purchased as single strands from IDT and annealed in lab.
Gibson Assembly Cloning
The constitutive reporters and dhdR constructs were produced using Gibson Assembly. The workflow for plasmid generation is shown below:
This was performed using Q5 master mix (NEB, cat. #M0491S).
This was performed using Dpn1 enzyme (NEB, cat. #R0176S).
This was performed using Monarch PCR & DNA Cleanup Kit (NEB, cat #T1030S) as well as DNA Clean and Concentrator (Zymo Biosciences, cat. #D4013).
This was performed using Zymoclean Gel DNA Recovery Kit (Zymo Biosciences, cat. #D4007).
This was performed using homemade Gibson master mix, which included DNA polymerase, DNA ligase, and 5’ endonuclease in buffer solution.
Restriction Enzyme Cloning
The binding site plasmids, both in pcDNA5 and constitutive reporter, were produced using restriction enzyme cloning.
This was performed using T4 DNA Ligase Reaction Buffer (NEB, cat. #B0202S) and T4 PNK (NEB, cat. #M0201S).
This was performed using the restriction enzymes NheI HF (NEB, cat. #R3131S) and AflIII (NEB, cat. #R0541S). Additionally, the reaction was carried out in rCutSmart buffer (NEB, cat. #B6004S).
This was performed using Quick Ligase (NEB, cat. #M2200S) in Quickligation Buffer (NEB, cat. #B6058S).
Bacterial Work
For transformations, we utilized Stable 3 cells (NEB, C3040H) and 5-alpha cells (NEB, C2988J). Agar plates were made with carbenicillin and homemade LB agar.
For inoculations, the media used was liquid LB broth with carbenicillin.
For minipreps, we utilized several different kits, including Zyppy plasmid miniprep kit (Zymo Biosciences, D4036), Zymo classic miniprep kit (Zymo Biosciences, D4054), and Monarch Plasmid Miniprep kit (NEB, T1010S).
Cell Work
This protocol outlines the maintenance of our three main cell lines, as well as cell counting and fixing. For passaging HEK and BXM cells, we utilized media made with DMEM (Gibco, 1193065), FBS (free sample provided by Gibco), and alpha-alpha (Gibco). Cell detachment required TrypLE (Gibco, 12605010) and PBS (Gibco, 10010002). Cell counting was performed with trypan blue (Gibco, 15250061).
For lipofections, two different forms of reagent were used. For standard HEK transfections, we used Lipofectamine 2000 (Thermo, 11668019) and Optimem (Gibco, 31985070). For non-standard BXM transfections, we used Lipofectamine LTX with PLUS reagent (Thermo, 15338100) and Optimem (Gibco, 31985070).
For this assay, the Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher, 16170) was used.
Stem Cell Protocol
hiPSC culture
Green fluorescent protein (GFP) labeled human induced pluripotent stem cells (hiPSC) was obtained at passage 2. The iPSCs were seeded onto pre-coated six-well plates using iPSC Coating Solution (Angio-Proteomie, cat. No. cAP-50). Media change was done daily with human iPSC culture medium (Xeno-free, serum-free, stable; Angio-Proteomie, cat. No. cAP-49). The cells were passaged at around 80% or higher confluency. Essentially, media was removed from the plate and 1 mL iPSC non-enzymatic dissociation solution (Angio-Proteomie, cAP-51) was added. The plate was then incubated at 37 °C for 5 minutes and the colonies were collected by pipetting up and down 3 times. The dissociation solution was removed with centrifugation at 200 g for 5 minutes, and iPSCs were seeded into pre-warmed iPSC culture medium with 10 µM ROCK inhibitor (Y-27632, Selleckchem-S1049). The iPSC cultures were incubated at 37 °C in 5% CO2. iPSC quality was examined using the Human Pluripotent Stem Cell Naive State qPCR Array (STEMCELL cat. No.07523) and was determined to be at prime state for differentiation.
The generation of cerebral organoids using the microfluidics system
Mainly, we followed the protocol of the STEMdiff Cerebral Organoid Kit (cat. #08570), with slight modifications to some steps. hiPSC-containing droplets were generated on day 0 using a microfluidics machine, with a relatively high cell density in each droplet (e.g. 100 cell/droplet). In particular, the droplets were made from 30% volume cell+media suspension in 70% Matrigel to provide structural support for the growth of the cells.The droplets were harvested and seeded into an Ultra-low Adherent 6 well plate for Suspension Culture (Stemcell, cat. #38071) in 2-3 mL EB Seeding Medium. Each well should contain around 2000-3000 droplets. Proper amounts of media were added on day 2 and 4. Upon progression into the induction and differentiation stages (day 5 and 7, respectively), old media was removed with the usage of a cell strainer and new media was dispensed. The ECM-embedding step on day 7 according to the original protocol was omitted as the EBs should develop inside the Matrigel droplet. Supplement all media with 0.5-1% Matrigel if EBs start to expand outside the droplet surface and fusion occurs. Finally, on day 10, the organoids were plated in maturation media as they enter the maturing stage; the use of an orbital shaker is optional as it facilitates organoid fusion. Perform media change every 3-4 days for the organoids until they mature after day 40 and are ready for downstream analysis or drug assays.
Immunofluorescent staining
Organoids were fixed in 4% paraformaldehyde overnight at 4 °C and allowed to sink in 30% sucrose overnight, followed by rapid freezing in Tissue-tek OCT Compound (Sakura Finetek #4583). Organoids were cryosectioned with a thickness of 10 µm. To stain, the sections were blocked in 0.25% Triton X-100 in TBS at 4 °C overnight. Primary antibodies were incubated with the sections with 10% horse serum and 1% BSA overnight at 4 °C, followed by secondary antibody incubation at the same conditions. The primary antibodies were diluted with the following dilutions: TUJ1 (BioLegend MMS-435P, 1:750), FOXG1 (Abcam ab196868, 1:200), PAX6 (DSHB, 1:200), FZD9 (OriGene TA344067, 1:200), GBX2 (Santa Cruz sc-81963, 1:200), PAX2 (Abnova M01, 1:200), SOX2 (Chemicon, AB5603, 1:300). Secondary antibodies were donkey Alexa Fluor 488 and 594. Nucleus was stained with DAPI.
Establishing a glioma and mini brain co-culture system for drug screening
For the co-culture system, we propose a general protocol here for future co-culture experiments.
- Briefly, a 96-well ultra-low attachment U-bottom plate is used to set up the experiment.
- The plate should include positive and negative control wells, i.e. mini brain only and glioma cell only conditions, with at least 3 replicates for each to avoid within group variations.
- Use cerebral organoid media in the co-culture system to ensure best performance.
- A low density of glioma cells should be seeded into each well. We recommend the amount of 500 cells/well in consideration of the small volume of the well. If the cancer cell line is fast-growing or drug-resistant, decrease seeding density.
- Add an equal amount of mini brains in each well. As a U-bottom plate is used, the mini brains will automatically collect to the bottom of the well and should be ideal for imaging purposes.
- Observe the plate over the course of 3-5 days. Take images regularly to document invasion behavior.
Downstream: The plate can be dosed with different drugs for screening purposes:
- We recommend having at least 3 titrations of concentration for each drug tested to observe both the killing effect and the potential toxicity of the drug on normal brain cells.
- The drug and the glioma cells should be dosed at the same time on day 0 into the co-culture system.
- Set up control wells where mini brains are dosed with glioma cells but no drug. They monitor the invasiveness of the glioma cells, and if no invasion is observed, the other wells stand no significant meaning.
- Observe over the course of 3-5 days and take images regularly to document the number and status of cells, migrations, and invasion.