Name-Surname: Aria Foroughi

ID: 2531465

CELL CULTURE AND HUMAN CYTOGENETICS

Aim: The study of chromosome structure and normal and abnormal functions (functions in
condensation, recombination, organization, and gene regulation) is the goal of cytogenetics.
Furthermore, the goal of cell culture is to create an environment that allows for maximum cell
proliferation, which is mainly achieved by the incubator (temperature, humidity, O2 and CO2
tension) and the basal cell culture medium and its supplements.

Introduction: Cell culture is the process of growing cells in a closed environment, usually
outside their native habitat. After cells of interest are isolated from living tissue, they can be
stored in a tightly monitored environment. In the exploration of basic scientific and
translational research concerns, cell culture is a very flexible technique. The use of cell lines
in scientific research is advantageous because of their homogeneity and attendant data
reproducibility. Cell cultures are used as model systems to study basic cell biology and
biochemistry, as well as interactions between cells and disease-causing factors such as
bacteria and viruses, drug effects, aging processes, and age-related triggers (Scott, 2011).

Materials and Methods:

Cell culture

1 preparation: Divide the medium into two T75 flasks in aliquots, giving each flask
12mL of medium. Fill each flask with 1mL MCF-7 cell line stock.

2 Harvesting: Remove supernatant and leave 0.5 mL liquid on cell pellet after
centrifugation at 400g for 8 minutes. Re-suspend the cells gently. By inverting the
mixture gently, add 6 mL of 0.075 M KCL 2 mL at a time. In a water bath, incubate the
tube at 37°C for 10 minutes. Remove supernatant after centrifuging cells at 400g for 8
minutes at room temperature. (At this point, the cells are swollen and fragile; treat with
care.) we use Kcl during this part since treatment with a hypotonic saline solution to
enhance cell volume is a crucial step in cell harvesting for chromosomal karyotyping.
Hypotonic solutions function by establishing a concentration gradient across the
cytoplasmic membrane, which causes water to rush in via active transport.

3 Fixation: Produce a 1:3 acetic acid: methanol fixative that is ice cold. 2 mL at a time,
add 8 mL cold fixative to the pellet with thorough mixing. Incubate the mixture for 20
minutes on ice. Remove supernatant after centrifuging cells at 400g for 10 minutes at
room temperature. 2 mL at a time, add 6 mL cold fixative to the pellet with thorough
stirring. Incubate the mixture for 20 minutes on ice. Eliminate supernatant after
centrifuging cells at 400g for 10 minutes at room temperature. Apply 0.5 mL of fixative
to the pellet. Pipette the pellet back into the 0.5 mL fixative solution. We use acetic acid
during fixation since It's utilized in compound fixatives to help avoid nucleic acid loss
and, because it swells collagen, to compensate for shrinkage caused by other
components like ethanol.
 4 Preparation of chromosome spread: Dip alcohol-cleared slides into 70 percent
methanol that is freezing cold. Let liquid to flow off the slide but do not pat it dry. Drop
the cell suspension using a pipette held at least 60 cm well above slide at a 45 degree
angle to the ground. Drops should be placed at the top of slides so that the cell solution
falls down and covers the whole surface. Pass rapidly crosses over the Bunsen
burner's flame. Place the specimens in a 37°C incubator to dry thoroughly. Label them
and keep them for one week at room temperature.

Cytogenetics
1. Warm phosphate buffer, which helps to maintain a constant PH in human
body, (0.025M, pH 6.8) in a 60°C water bath for 10 minutes.
2. For 10 minutes, soak the dry slides in warm PBS.
3. 6.25 mL phosphate buffer, 0.25 mL 1 percent trypsin solution, 2.5 mL
methanol, and 1 mL stock toxic Giemsa stain = 10 mL working Giemsa stain.
4. Place treated slides in a petri dish and cover with working Giemsa solution;
incubate at room temperature for 20 minutes.
5. Detach forcepts from slides and wash with tap water. Leave for total air
drying.
6. Evaluate the nuclear material of the cells whose contents have been evenly
disseminated over the slide surface using a 100X objective (oil immersion).

Results:

•telocentric (no p arm, centromere is at the end)

•acrocentric (very small p arm, centromere is close to the end)
•submetacentric (p arm is just a little smaller than q arm, centromere is close to the middle),
•metacentric (p and q arms are the same length, centromere is at the middle of
chromosome).
Giemsa stained MCF-7 human breast cancer cell sister chromatids

23 pairs of Giemsa-stained human chromosomes under 100X magnification

Giemsa stained MCF-7 human breast cancer empty cell (with no chromosomes under 100X
magnification.

Discussion:
1 MCF-7 is a breast cancer cell line that was discovered in a 69-year-old White
woman in 1970. MCF-7 stands for Michigan Cancer
Foundation-7 Herbert Soule and colleagues first created cell lines in 1973 at their institute in
Detroit.
- MCF-7 cells have epithelial markers including E. cadherin, -catenin, and Cytokeratin 18
(CK18) but
Not mesenchymal markers like vimentin and smooth muscle actin, indicating that they are
differentiated mammary epithelial cells (SMA).
- MCF-7 cells are excellent for in vitro breast investigations because they preserve certain
perfect mammary epithelial features such asestrogen processing in the form of estradiol via
estrogen receptors (ER) in the cell cytoplasm.It'sthe first breast cancer cell line to respond to
hormones(Comşa et al., 2015).
2 After a period of cell development and proliferation, the injection of colchicine,
which poisons the mitotic spindle, causes dividing cells to enter metaphase. The
cells were subsequently exposed to a hypotonic solution, which caused their
nuclei to enlarge and burst. Colchicine has a harmful impact on live cells, causing
the process of mitosis to be halted at an early stage, generally the metaphase,
with the appearance of strange and irregular nuclear configurations, and
frequently cell death. Colchicine also blocks spindle formation and terminates
metaphase cells when it binds to tubulin(O’Connor, 2008).

3 A karyotype test examines the size, shape, and number of chromosomes in your
body. This test is very helpful for the determination of any genetic disorder. New
methods of karyotyping have recently been introduced. The use of fluorescent
dyes is very useful. It binds to specific regions of chromosomes. When there is
the use of specific probes, which consist of various amounts of dyes, the different
pairs of chromosomes achieve a unique spectral activity. In this, the pairing which
occurs between the homologous chromosome is simpler because homologous
pairs consist of the same color. Spectral Karyotyping is been used to detect
translocation, which is not easily recognized by the traditional branding analysis.
When working closely with short tandem repeat analysis, copy number variation
sequencing is more accurate and reliable than karyotyping and can be used to
identify abnormalities in chromosomal copy numbers(Luminochem _ Water-
Soluble UV Fluorescent Dyes, n.d.).
The FISH, which Fluorescence in situ hybridization. It is a technique that is used in
the laboratory to locate and detect a specific DNA sequence on a chromosome. The
Disadvantage of FISH techniques is that it only detects the duplications and deletions of
regions, which are specifically targeted by the probe(Fluorescence In Situ Hybridization
(FISH) | Learn Science at Scitable, n.d.).

In some groups you observed just a few chromosome clusters while in others you had full
slides. Discuss what could have gone wrong for those "empty slides"; you need to go
through the whole procedure step by step and talk about the parts you could have
harmed/lost cells.
Discuss the figures, what do you see in some of the figures, why do you see these
structures, what could be the reason?
References
Comşa, Ş., Cîmpean, A. M., & Raica, M. (2015). The story of MCF-7 breast cancer
cell line: 40 Years of experience in research. In Anticancer Research (Vol. 35,
Issue 6, pp. 3147–3154).
Fluorescence In Situ Hybridization (FISH) | Learn Science at Scitable. (n.d.).
Luminochem _ Water-soluble UV fluorescent dyes. (n.d.).
O’Connor, C. (2008). Karyotyping | Learn Science at Scitable. In Nature Education
1(1) (p. 27). https://www.nature.com/scitable/topicpage/karyotyping-for-
chromosomal-abnormalities-298/%0Anature.com/scitable/topicpage/
karyotyping-for-chromosomal-abnormalities-298/
Scott, C. (2011). Cell culture. In BioProcess International (Vol. 9, Issue SUPPL. 9,
pp. 24–31).