Cell Culturing (Böttcher)

Question 1

Advantages of mouse-to-human translation

Answer 1

• Cheaper
• Easier to do manipulations
• More availability (can’t order human brains online, can only get from patients in the form of a biopsy or wait until a donor passes away and do an autopsy)
• Can have healthy Control
• Possibility for long term storage

Question 2

Limitations of brain biopsy

Answer 2

• Lack of regional specificity
• Must be a reason for biopsy, meaning the tissue is at least slightly pathological (i.e. brain tumor, from surgery for epilepsy, etc.)

Question 3

Limitation brain autopsy

Answer 3

• Most will pathogenic
• Will get healthy, but not really healthy tissue along pathology path (part of this is that brain needs to be harvested faster than is usually possible for autopsy)

Question 4

In vitro human tissue and cell culture models

Answer 4

• Primary cell culture
• Multiple/mixed cell type culture
• Organotypic brain slice culture
• Organoid culture
• iPSC-derived neuron/glia culture

Question 5

What to know for using cells to study brain function

Answer 5

• Cell type of interest
• Dynamic interaction(/other cell types of interest)
• Stimulation/disease condition
• Manipulation using pharmacological agents
• Etc.

Question 6

How to do a primary cell culture

Answer 6

1. Take biopsy or autopsy
2. Do cell suspension using flow cytometry or MACS
3. Culture cells using a system of medium, growth factors, and sampling (cells) in a well plate or flask

Question 7

MACS

Answer 7

Magnet-Activated Cell Sorting

• Cells not really activated when they’re separated
• Based on antibody interactions where antibodies will bind to antigen of interest (ex: CD11b are specific to microglia)
• Antigens/antibodies are bound to a magnetic labeled bead
• Cell suspension put into a column
• Column is placed into magnetic bar, creating a magnetic field
• Cells of interest remain in column and are flushed out at the end

Question 8

FACS/flow cytometry

Answer 8

Fluorescence-associated Cell Sorting
- Antibody is conjugated with a fluorescence dye, allowing for selection of cells that are positive for a particular antigen using a fluorescence detector

Question 9

Limitations to primary cell culture

Answer 9

• Short lifespan (taking cells in the brain from a highly complex environment into a simple culture environment leads to shortened lifespan)
• Cells tend to have artefacts
• Potential for changes in phenotype and function (due to change in environmental cues)
• Inability to see dynamic interactions
• Less or no proliferative capability
• Cryopreservation-induced cell death (will lose many cells during course of freeze-thaw cycle)

Question 10

Parameters to be considered for organotypic brain slice culture

Answer 10

• Thickness (usually around 300 um, but can vary depending on type of analysis you want to do later)
• Well plate, flask, or insert? (usually use insert because organotypic brain slice cultures require high oxygen concentrations and the insert allows for good oxygen exchange)
• Medium
• Growth Factors (Do you need extras? For example, microglia need TGF-b to keep function in culture, otherwise will become macrophages)
• What kind of analysis do you want to do later?

Question 11

Limitations of organotypic brain slice culture

Answer 11

• Short life
• Artefacts
• Blood monocyte/myeloid cell contamination → tissue macrophage
• Cryopreservation-induced cell death

Question 12

How to get induced pluripotent stem cells (iPSCs)

Answer 12

1. Take primary cells (like patient-specific fibroblasts, blood cells, or during cells)
2. Reprogram cells
3. Culture/differentiate reprogrammed cells into cells of interest (ex: astrocytes, microglia, hematopoietic progenitor cells, etc.)

Question 13

Potential uses of iPSCs

Answer 13

• Could perform a 3D in vitro human brain model with a functional BBB
• Could use for drug discovery
• Could be used therapeutically, if cells can be re-introduced into patient

Question 14

How to obtain organoid culture

Answer 14

1. Derive from iPSCs
   - ESCs/iPSCs → floating spheroids → culture
2. Directly derive organoids
   - harvest tissue/obtain tissue biopsy → dissociate into functional units → enrich for stem cells

Question 15

What does organoid culture allow us to do?

Answer 15

• Omics profiling
• Study host-microbe interaction
• Gene editing → targeted corrections of mutations or disease modeling
• High throughput drug screening

Question 16

Limitations of iPSCs and organoid culture

Answer 16

1. Maturation
   - Can generate microglia, neurons, etc. from iPSCs or organoid culture, but whether they’re fully functional is still debated
   - Back-transplanted iPSC neurons to brain don’t proliferate/differentiate properly → not fully mature yet?
2. Tissue complexity/heterogeneity
   - Could lead to heterogeneity of result culture
   - Could find different cells in organoid culture
   - Could be same cell w/ different states or different cells in the culture
   → need to check for culture purity first
3. High variation between clones (culture not really stable)
4. Chromosomal abnormalities (particularly in long-term culture) have tumorigenic qualities

Question 17

In vitro analytical techniques for medium

Answer 17

Western Blot
ELISA
Multiplexed Elisa
MS
HPLC

Question 18

in vitro analytical techniques for/in cells

Answer 18

1. Seahorse
   - Gives info about metabolism about cell of interest (mitochondria function)
   - Use well plates w/ electrodes in each well
2. CyTOF (mass cytometry)
   - = a variation of FACS where antibodies are labeled w/ heavy metal ion tags rather than fluorochrome
   - Allows for combination of many more antibody specificities in a single sample
   - Is high throughput
3. Flow Cytometry/FACs
4. RT-PCR
   - = reversible transcription PCR
   - Uses RNA as template
5. scRNA-Seq
   - Biopsy taken, then FACS is done where w/ 1 cell per well
   - Followed by sequencing, unbiased clustering, and identification of subsets
   - Each individual cell is sequenced and results can be further validated by ICH and CyTOF
   - scRNA-Seq + CyTOF gives minimal signal spillover to neighboring metals/cells, which is important if you’re studying cell signaling pathways or interactions between cell types that go through signaling pathways
   - Can do maximum 10-20k cells at once at around 4000 euro per sample

Question 19

In vitro analysis of tissue

Answer 19

(immuno)Histology

Imaging CyTOF

Question 20

Result interpretation of in vitro models

Answer 20

• Advanced statistical testing (like biological replications, identification of confounders, etc.)
• Is in vitro to in vivo translation plausible? Does it work? (might have to do reverse translational research; VERY IMPORTANT TO INCREASE VALIDITY)
• Experimental confirmation/evaluation of results obtained from in vitro study (ex: reverse translational research)

-Longitudinal or snapshot analysis (what you see in culture is just a snapshot in time obtained from one patient at one particular time point from one particular region of the brain; = snapshot in space and time)
→ longitudinal studies possible in rare cases (like with iPSCs), but it’s difficult to do longitudinal studies that are also representative of longitudinal studies of patients