Group A

Haematopoiesis established by 2D and 3D

cultures and their assays
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 Nusra- leader

Amro- coordinator

Members and Zubaida- Liaison

their roles Jeorgia- Recorder

Mustafa- Evaluator

Maria- Spokes person

 Introduction

2D cell cultures

3D cell cultures
Key contents Prospective use

Haematopoiesis assays

Cost of investigating cell cultures in the lab

What is Haematopoiesis?

• Is the synthesis of blood

cell components. It takes
place during embryonic
development and
throughout maturity,
replenishing the blood
system.

Image taken from

https://app.pulsenotes.com/medicine/haematology/notes/haemat
opoiesis
 Hematopoiesis in-vitro
investigations began, scientists
wanted to investigate the
viability of cultivating blood
cells outside the body.
Erythropoietin (hormone that
stimulates the production of red
blood cells) was discovered

1900s 1960s

History
1940s Currently

Scientists started using scientist perform in-vitro

specialized media with growth
factors and hormones to
encourage the development and
differentiation of HSCs.
Hematopoiesis studies to learn
more about the origins of blood
cells and to discover treatments
for blood disorders such as
sickle cell disease, lymphoma
and leaukaemia.
2D cell culture

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 Introduction
• 2D cell culture refers to a laboratory technique in which cells are
grown and maintained on a flat surface, such as the bottom of a
culture dish or a glass slide (Dornhof et al., 2022).
• In 2D cell culture, cells are typically grown in a nutrient-rich
medium and kept at a controlled temperature and humidity to
promote growth and survival.
• The cells can be used for a wide range of applications, such as
studying cell morphology, proliferation, differentiation, and drug
screening (Russo et al., 2022).
• As a result, 2D culture may not accurately mimic the complex
biology of tissues and organs in the body, and may not always
provide reliable results.
 Uses of 2D cell culture in Hematopoiesis
● Used to maintain Hematopoietic stem ●
cell in vitro and study their self-
renewal, differentiation, and lineage
commitment (Liu et al., 2022).
● Used to induce the differentiation of
hematopoietic progenitor cells into
specific blood cell lineages, such as
erythrocytes, leukocytes, and
platelets.
● .
2D culture systems can be used for
high-throughput screening of drugs
or compounds that affect blood cell
development or function which helps
to identify potential therapies for
blood disorders or drugs that have
adverse effects on blood cell function
(Torres-Quesada et al., 2022).
● Used to model blood disorders such
as leukemia and lymphoma.
 Pros and Cons of 2D Cell Culture
Pros
• 2D cell culture is a relatively
simple and low-cost method for
growing and maintaining cells in
the laboratory (Russo et al.,
2022).
• Cells can be easily observed and
analysed under a microscope,
allowing for detailed
characterization and
quantification of cell behaviour
(Engel et al., 2022).
Cons
• 2D cell culture lacks the complexity of
the 3D microenvironment found in vivo,
which can limit the accuracy and
relevance of the results obtained.
• Cells in 2D culture may exhibit different
phenotypic and functional properties
compared to cells in vivo, which can
affect the interpretation of experimental
data (Torres-Quesada et al., 2022).
 Pros and Cons of 2D Cell Culture
Pros
Cons
• The lack of physical support structures
• It is a well-established technique, and gradients in 2D culture can limit the
with standardized protocols and ability to study cell-cell interactions,
reagents available for a wide tissue architecture, and mechanical
range of cell types. transduction.
• Some cell types may not grow or
differentiate properly in 2D culture and
may require more complex 3D culture
systems.
 3D Cell Culture
• A 3D cell culture is an artificially created environment in which biological
cells are permitted to grow or interact with their surroundings in all three
dimensions. Unlike 2D environments, a 3D cell culture allows cells in
vitro to grow in all directions, similar to how they would in vivo.
• Types of 3D cultures are
• Spheroid culture.: Microchips, embryoid bodies, collagen gels and
hanging-drop culture
• Suspension cultures on non-adherent plates.
• Cultures in concentrated medium or in gel-like substances

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 Uses of 3D cultures in Hematology
• Spheroids can be used to study the process of cell migration on ECM
proteins, invasion into Matrigel, or simultaneously tissue invasion and
angiogenesis. Characteristic features of migration are visible flattened
cells surrounding spheroids. (Kapałczyńska et al 2018)

• Moreover, 3D tissue culture system allows for the creation of imitation

cancer tissue, with green fluorescent protein (GFP) expression and
with the features of a solid tumour. (Kapałczyńska et al 2018)

• Apart from using 3D systems in the area of cancer research, they can
also be applied in tissue engineering. (Kapałczyńska et al 2018)

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 Pros and cons of 3D culture
(Kapałczyńska et al 2018)
• 3D cultures like soft agar
allows to study both the
growth of a single cell
regardless of attachment.
• Cells in Matrigel have three-
dimensional interactions with
the local environment and
form tissue-like structures
• Used to study the
aggressiveness of the cells and
their potential for metastasis
• It requires the separation of single
cells from spheroid structures by
proteolytic degradation of single
layers.
• In many 3D methods, the efficiency,
life-span, repeatability, and comfort of
work are poorer than in the case of
2D systems
• “spheres” can be formed, not from a
single cell, but from a few cell
clusters.
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 Study: Allogeneic cell therapy using umbilical cord MSCs on collagen
scaffolds for patients with recurrent uterine adhesion: a phase I clinical trial.
Trials: (Cao et al 2018)
(NCT02313415). Trial name: Treatment of Infertility by Collagen Scaffold
Loaded with Umbilical Cord Derived Mesenchymal Stem Cells.10th
December 2014
Results showed that
The intrauterine adhesion score decreased and endometrial thickness in
patients increased.
Mesenchymal stem cells from umbilical cord differentiated into patient’s
cells. DNA from the uterine wall was the patients DNA. (Cao et al 2018)

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 2D cell culture vs 3D cell culture
• In 2D cell culture, cells are grown on a flat surface, while in 3D cell culture, cells are
grown in a three-dimensional environment that better mimics the natural architecture of
tissues and organs (Engel et al., 2022).
• In 3D culture, cells are exposed to gradients of nutrients and oxygen, which can impact
their behavior and function in ways that are not observed in 2D culture (Russo et al.,
2022).
• 2D culture is generally easier and faster to perform, while 3D culture can be more
complex and time-consuming.
Prospective in-vitro cell culture studies
In haematopoiesis, these studies identify the type of blood cell
haematopoietic stem cells (HSCs) will develop into before it
happens
Prospective identification: Molecular markers on the surface of
HSCs are used by flow cytometry to screen the cell population
needed
Prospective isolation: Fluorescence activated cell sorting
purifies the population, removing it from other HSC types
Prospective in-vitro studies are needed because:
• More functional HSCs = mature blood cells to be produced,
almost endlessly
(Schroeder, 2010; Che et al., 2022; Bleichrodt and Read, 2019, p.3, fig. 1)
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Prospective in-vitro cell culture studies
• Improved isolation techniques = higher purity in HSCs
populations
• Previously unfeasible techniques can now be carried out e.g.
global proteomics, metabolomics, and ChIP-sequencing, small
molecules and CRISPR
• More functional HSCs = maintain their function throughout
gene manipulation,

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Study one – novel markers
The aim was to see if functional mouse HSCs could be
distinguished with novel in vitro molecular markers - Fgd5 or
ESAM in conjunction with EPCR
HSC clones were taken from a single-cell culture expansion after
10 and 28 days for prospective identification and isolation and
analysed in comparison to conventional markers.
It was found that:
• In short and long term cultures, prospective identification and
isolation with these markers was reliable whether Fgd5 or ESAM
was used
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Study one – novel markers
• Molecular profiling using RNA sequencing showed
HSCs in culture expansion multiply like foetal HSCs
and behave like freshly isolated HSCs

• (Che et al., 2022; Che et al., 2022, p. e fig. 2)

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 Study two – novel culture technique
The aim was to preserve functional long term HSCs without proliferation using a
novel 7-day HSC hibernation culture technique
Two mediums of IL-11 with or without serum were used to culture single HSCs
for 7 days.
These HSCs were then cultured in cytokine-rich methylcellulose colony forming
cell assays for 14 more days
They were then used for prospective identification and isolation using molecular
marker CD150 and analysed in comparison to freshly isolated HSC.

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It was found that:
• The single HSCs remained functional during hibernation without proliferation
• Prospective identification and isolation with CD150 was reliable
• Molecular profiling using RNA sequencing showed HSCs in colony forming
cell assay behave like freshly isolated HSCs

(Oedekoven et al., 2021; Oedekoven et al., 2021 1619. fig. 4.)

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Assays
• Biological assays are experimental methods for assessing the presence,
localization, or biological activity of a substance in living cells and
biological matrices. Such methods are essential to biological science
and technology.

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Flow cytometry
Advantages
High speed detection of cells depending on Expensive and training needs to be
the flow rate.
The input of the cells can vary, and many
cells can be measured simultaneously.
Small colonies of cells can be identified.
Sorting rates about 100,000 cells per
second
The equipment used is portable.
Can be available on 3 PMT or 5 PMT
Disadvantages
done to operate the instruments.
Some flowcytometry use two or more
lasers which could lead to errors.
Cleaning needs to be done as
blockages could lead to back logs.
Cells need to be manually fed to the
flow cytometer resulting in the loss of
the cell culture if errors are to be
made.
Reduction of sensitivity due to the use
of multichannel PMT
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 Histological staining
Advantages Disadvantages
Better visualisation of cell structures Staining had to be done by hand and is
not automatic.

Cheap Addition of fixatives can lead to poor

staining.
The activity of cells can be Requires labour and time
demonstrated
Staining can be done on cover slips Different stains are available for different
cells.

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 Costs of running and maintaining 2d and 3d cell cultures

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040128/
• Costs of setting up and maintaining a 2D in-vitro cell culture is relatively
inexpensive, commercially available (both tests and the media), and are more
commonplace than 3D cell cultures
• 3D cell cultures are much more expensive, are more time-consuming, and have
fewer commercially available tests, as it’s a much newer technology.
• In Suspension cultures on non-adherent plates, some cell lines need expensive
plates coated with specific materials (polystyrene, covanlently bound
hydrogel).
• Costs are predicted to decrease due to improvements to automation and cost
reductions surrounding the technology, and will be predicted to replace 2D cell
cultures as it mimics in-vivo interactions due to the “niche” environment
created in the 3D cell culture – higher drive to make it mainstream and
convenient. 25
cell line cost

• Bone Marrow (BMMC) – £450

• Blood Cell lines (PBMC) - £300
• Human Embryonic Stem Cells (BG01V) - £1000
• Placenta cells (CRL-7526) - £800

All produce pricing was found and searched on ATCC:

• https://www.atcc.org

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Equipment costs
• https://www.nature.com/articles/sdata2017170
• 2D cell culture
• 96 well plate (Greiner Bio One #655-088) are between £250-400
• All major equipment are found commonly in a laboratory – no/few specific
equipment purchases needed.
• 3D cell culture
• 384 well plate (Greiner Bio-One #781090) - £3400
• 384 well spheroid plate hydrogel plate - £2500

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 References
• Dornhof, J., Kieninger, J., Muralidharan, H., Maurer, J., Urban, G.A. and Weltin, A.,
2022. Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in
3D cell cultures. Lab on a Chip, 22(2), pp.225-239.
• Engel, M., Belfiore, L., Aghaei, B. and Sutija, M., 2022. Enabling high throughput drug
discovery in 3D cell cultures through a novel bioprinting workflow. SLAS technology,
27(1), pp.32-38.
• Liu, B., Tao, C., Wu, Z., Yao, H. and Wang, D.A., 2022. Engineering strategies to
achieve efficient in vitro expansion of haematopoietic stem cells: development and
improvement. Journal of Materials Chemistry B, 10(11), pp.1734-1753.
• Russo, M., Cejas, C.M. and Pitingolo, G., 2022. Advances in microfluidic 3D cell
culture for preclinical drug development. Progress in Molecular Biology and
Translational Science, 187(1), pp.163-204.
• Torres-Quesada, O., Doerrier, C., Strich, S., Gnaiger, E. and Stefan, E., 2022.
Physiological Cell Culture Media Tune Mitochondrial Bioenergetics and Drug
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Sensitivity in Cancer Cell Models. Cancers, 14(16), p.3917.
Hristu, R., Stanciu, S.G., Dumitru, A., Paun, B., Floroiu, I., Costache,
M. and Stanciu, G.A., 2021. Influence of hematoxylin and eosin
staining on the quantitative analysis of second harmonic generation
imaging of fixed tissue sections. Biomedical Optics Express, 12(9),
p.5829.

Robinson, J.P., 2004. Flow cytometry. Encyclopedia of biomaterials

and biomedical engineering, 3, pp.630-642.
Front page images:
(2021), Sanquin, Available at:
https://www.sanquin.org/news/2021/jun/large-boost-for-sanquin-resear
chers-to-culture-blood-cells#
, (16/03/23)
Admin,K.(2019), Kosheeka, Available at:
https://kosheeka.com/5-tips-for-successful-animal-cell-culture-experien 29
• Cao, Y., Sun, H., Zhu, H., Zhu, X., Tang, X., Yan, G., Wang, J., Bai, D., Wang, J.,
Wang, L. and Zhou, Q., 2018. Allogeneic cell therapy using umbilical cord MSCs
on collagen scaffolds for patients with recurrent uterine adhesion: a phase I
clinical trial. Stem cell research & therapy, 9(1), pp.1-10.
• Mezey, É., 2016. On the origin of blood cells-Hematopoiesis
revisited. Oral diseases, 22(4), p.247
• Kapałczyńska, M., Kolenda, T., Przybyła, W., Zajączkowska, M.,
Teresiak, A., Filas, V., Ibbs, M., Bliźniak, R., Łuczewski, Ł. and
Lamperska, K., 2018. 2D and 3D cell cultures–a comparison of
different types of cancer cell cultures. Archives of Medical
Science, 14(4), pp.910-919.

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