NAME: Aman Paul

Date: 13 Feb 2023

Year and Section: BS in Biology

GENERAL DIRECTIONS: Read each question carefully. Review your answers before submitting them.
Part 1 Outline the general steps involved in the following processes.
1. Disaggregation of Tissue:
 Obtain tissue sample.
 Wash the tissue to remove debris and blood.
 Mechanical disaggregation: mincing or chopping the tissue into smaller pieces.
 Chemical disaggregation: enzymatic digestion using proteolytic enzymes such as trypsin or collagenase to break down
extracellular matrix.
 Filter the disaggregated tissue to obtain single-cell suspension.
 Centrifugation to pellet cells and remove excess solution.
2. Cell Count and Viability:
 Stain cells with a dye that distinguishes live from dead cells (e.g., trypan blue).
 Prepare a hemocytometer or an automated cell counter.
 Load a sample of the cell suspension onto the counting chamber.
 Under a microscope, count the number of live and dead cells in the grid squares.
 Calculate the cell concentration and viability using appropriate formulas.
3. Cell Freezing:
 Prepare a cryoprotectant solution (e.g., dimethyl sulfoxide or glycerol) to protect cells during freezing.
 Mix the cell suspension with the cryoprotectant solution.
 Transfer the cell-cryoprotectant mixture into cryovials or cryotubes.
 Gradually cool the cells using a controlled-rate freezer or a freezing container in a -80°C freezer.
 Transfer the cryovials into liquid nitrogen for long-term storage.
4. Cell Thawing:
 Retrieve cryovials containing frozen cells from liquid nitrogen.
 Quickly thaw the cryovials in a water bath or under running warm water until just a small ice crystal remains.
 Transfer the thawed cells into a pre-warmed culture medium or buffer.
 Centrifuge the cell suspension to pellet the cells and remove the cryoprotectant.
 Resuspend the cells in fresh medium and plate them for further experimentation or culture.

Part 2 Research applications

Look for five research articles that use cell isolation and cell culture as part of their research methodologies.
Identify the cell line used and the methods of cell isolation and cell culture adapted in each research.

Journal Article 1
Title: Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white
adipocytes to analyze cell-cell communication
Authors: Vaishali Chaurasiya, Dan Duc Pham, Jukka Harju, Anne Juuti, Anne Penttilä, Sharath Kumar Goud
Emmagouni, Van Dien Nguyen, Birong Zhang, Sanni Perttunen, Salla Keskitalo, You Zhou, Kirsi H. Pietiläinen, P.A.
Nidhina Haridas, Vesa M. Olkkonen
Type of cell and cell-line: Human visceral adipose tissue microvascular endothelial cells (AMvEC)
Cell isolation and cell culture methods: The authors developed a method for the isolation of adipose microvascular
endothelial cells (AMvEC) from visceral adipose tissue (VAT) biopsies of subjects with obesity. Additionally, mature
white adipocytes were isolated from the VAT biopsies by adapting a previously published Membrane aggregate adipocytes
culture (MAAC) protocol. The identity and functionality of the cultivated and isolated AMvEC were validated through
various methods including imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting
(FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, the authors
established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for
the analysis of communication between the two cell types. The communication between endothelial cells and adipocytes
was validated by omics analyses, revealing alterations in various proteins and pathways involved in metabolism,
intracellular transport, and signal transduction. This co-culture system could be employed for studying cell-cell
communication and drug screening.
Reference: Chaurasiya, V., Pham, D. D., Harju, J., Juuti, A., Penttilä, A., Emmagouni, S. K. G., ... Olkkonen, V. M.
(2023). Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white
adipocytes to analyze cell-cell communication. Experimental Cell Research, 405(2), 113819.
https://www.sciencedirect.com/science/article/abs/pii/S0014482723003701?via%3Dihub

Journal Article 2
Title: Isolation and profiling of viable tumor cells from human ex vivo glioblastoma cultures through single-cell
transcriptomics
Authors: Junyi Zhang, Jakob Straehle, Kevin Joseph, Nicolas Neidert, Simon Behringer, Jonathan Göldner, Andreas
Vlachos, Marco Prinz, Christian Fung, Jürgen Beck, Oliver Schnell, Dieter Henrik Heiland, Vidhya M. Ravi
Type of cell and cell-line: Tumor cells from human glioblastoma cultures
Cell isolation and cell culture methods: The researchers took tissue samples from brain tumors and cut them into small
pieces. They grew these tissue pieces in a special liquid for two days. Then, they put tumor cells onto these tissue pieces
where the gray and white parts of the brain meet. After 7-9 days, they broke down the tissue into individual cells. Using a
method called fluorescence-activated cell sorting (FACS), they separated the tumor cells that glowed green (because they
were marked with a special protein called ZsGreen) from the rest of the cells. Finally, they looked at the genes expressed
in each of these isolated tumor cells to learn more about them.
Refrence: Zhang, Junyi, et al. “Isolation and Profiling of Viable Tumor Cells from Human Ex Vivo Glioblastoma
Cultures through Single-Cell Transcriptomics.” STAR Protocols, vol. 4, no. 3, 15 Sept. 2023, p. 102383,
https://www.sciencedirect.com/science/article/pii/S2666166723003507

Journal Article 3
Title: Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined
serum-free culture conditions
Authors: Takayuki Fukuda, Kazuo Takayama, Mitsuhi Hirata, Yu-Jung Liu, Kana Yanagihara, Mika Suga, Hiroyuki
Mizuguchi, Miho K. Furue
Type of cell and cell-line: Human pluripotent stem cell-derived hepatic progenitor cells
Cell isolation and cell culture methods: The authors developed a new serum-free culture condition for isolating and
expanding human pluripotent stem cell-derived hepatic progenitor cells. This method uses growth factors and small
molecules to promote the differentiation of human pluripotent stem cells into hepatocyte-like cells through a hepatoblast-
like stage.
Refrence: Fukuda, Takayuki, et al. “Isolation and Expansion of Human Pluripotent Stem Cell-Derived Hepatic Progenitor
Cells by Growth Factor Defined Serum-Free Culture Conditions.” Experimental Cell Research, vol. 352, no. 2, 1 Mar.
2017, pp. 333–345, https://www.sciencedirect.com/science/article/pii/S0014482717300757 .

Journal Article 4
Title: Isolation and culture of mouse alveolar type II cells to study type II to type I cell differentiation
Authors: Qian Chen, Yuru Liu
Type of cell and cell-line: Mouse alveolar type II cells
Cell isolation and cell culture methods: The article describes a method for isolating and culturing mouse lung alveolar
type II cells. Briefly, the lungs are perfused with PBS and instilled with dispase to loosen the cells. The tissue is then
dissociated with DNase I and the cells are filtered to obtain a single-cell suspension. The type II cells are then enriched by
magnetic beads sorting or flow cytometry sorting. For 2D culture, the cells are plated on tissue culture plates coated with
CD45 and CD32 antibodies. For 3D culture, the cells are co-cultured with Mlg cells in Transwell inserts. Both 2D and 3D
cultures can be treated with S1P or JTE-013 to study the effects of these factors on type II cell differentiation.
Refrence: Chen, Qian, and Yuru Liu. “Isolation and Culture of Mouse Alveolar Type II Cells to Study Type II to Type I
Cell Differentiation.” STAR Protocols, vol. 2, no. 1, Mar. 2021, p. 100241,
https://www.sciencedirect.com/science/article/pii/S2666166720302288

Journal Article 5
Title: Isolation of Toxoplasma gondii in cell culture: an alternative to bioassay
Authors: Tania Dawant, Wei Wang, Maria Spriggs, Geraldo Magela de Faria Junior, Laura Horton, Nicole M. Szafranski,
Helga Waap, Pikka Jokelainen, Richard W. Gerhold, and Chunlei Su
Type of cell and cell-line: Vero cells and human foreskin fibroblast (HFF) cells
Cell isolation and cell culture methods: Heart tissue from highly infected animals is homogenized and treated with
enzymes to dissociate cells. This mixture is then directly inoculated onto Vero or HFF cells and incubated for 21 days.
Cultures are monitored for signs of T. gondii growth, especially tachyzoites. If negative, cells are passaged onto fresh
cultures and monitored again. Finally, T. gondii presence is confirmed with PCR or immunofluorescence assay. This cell
culture method offers a simpler alternative to traditional bioassays.
Refrence: Dawant, Tania, et al. “Isolation of Toxoplasma Gondii in Cell Culture: An Alternative to Bioassay.”
International Journal for Parasitology, 13 Dec. 2023,
https://www.sciencedirect.com/science/article/abs/pii/S002075192300228X

Conclusion:
1. According to your readings, what are the research applications of cell isolation and culture?
 Studying cell function and behavior: By isolating and culturing specific cell types under controlled
conditions, researchers can observe their growth, differentiation, and response to various stimuli in detail. This is
valuable for understanding various diseases, developing new drugs, and exploring fundamental biological processes.
 Generating new cells for transplantation: Culturing certain cell types like stem cells or progenitor cells provides a
potential source of cells for transplantation therapies in regenerative medicine.
 Drug discovery and screening: Cultured cells can be used to test the effects of new drugs and identify potential
candidates for further development.
2. How do cell isolation and cell culture methods vary in each research?
 Origin of cells: Each study utilizes different starting materials. Some isolate cells directly from tissues (e.g., tumors,
adipose tissue, lungs), while others use established cell lines or derive cells from pluripotent stem cells.
 Isolation techniques: Depending on the cell type and tissue source, researchers employ various methods like
enzymatic digestion, mechanical dissociation, or FACS sorting for isolation.
 Culture conditions: Each study utilizes specific culture media and protocols tailored to the needs of the isolated cells.
This includes factors like growth factors, temperature, oxygen levels, and co-culture with other cell types.
 Downstream analysis: Researchers use various techniques to analyze the isolated and cultured cells, including
microscopy, gene expression profiling, protein assays, and functional assays.