CRITICAL REVIEW ON HAEMATOPOIESIS

ESTABLISHED BY 2D AND 3D CULTURES AND

THEIR ASSAYS.
Lecturer

Dr. Amgad Gerges

Haematology

Msc. Biomedical Science

Student

Aminath Nusra Faiz

(22028548)

London Metropolitan University

 1. Introduction

This is a report is written as an assessment for the course work MSc. Haematology by
Biomedical Science students. The aim of this report is to research the history and recent
studies of 2D and 3D cell cultures and critically analyse them to answer the following points
 How they relate to normal physiology.
 How they helped our understanding of haematopoiesis.
 How they could be used prospectively.
 Which is the best assay.
 The advantages and disadvantages of various assays.
 Are there any limitations of these study.

History of cell cultures.

Cell culture involves the isolation and expansion of cells derived from animal, insect or plant
origin using favourable conditions provided in the laboratory. Establishing of cell cultures
was first done in the mid 1900s by Ross Harrison with the help of hanging drop method.
(Ham et al. 2019).

History of Haematopoiesis established in vitro.

History
1970 Establishment of 3D cultures of soft agar
solution by Hamburger and Salmon. (Ham et
al. 2019)
1989 and 1992 Coining of the term 3D culture by
Barcellos-Hoff and colleague and Petersen
et al. (Ham et al. 2019)

1990 Purification and transplantation of HSC by

Bensinger et al.1996, Shpall et al., 1999

(Mazey 2016)
2000 Use of Fluorescence Activated Cell Sorting
by Akashi et al, Kondo et al (Mazey 2016)

2D culture involves growing of cells on a single layer of agar mediums supplied with
chemicals on flat surfaces such as Petri dishes. Whereas 3D cultures involve growing of cells
on suspension cultures on unattached surfaces like gel medium, hydrogel scaffold cultures
and organoids. ( Kapałczyńska et al. 2018)
2. How the studies conducted in the field of haematology could be used
prospectively.

 Study: A simple and effective method to purify and activate T cells for successful
generation of chimeric antigen receptor T (CAR-T) cells from patients with high
monocyte count. (Wang et al 2022)

 The cell cultures are used to prospectively to identify the type of cells established in
the culture and to devise a treatment for the known diseases today. An ongoing
clinical trial for the use of CAR-T cells are mentioned as follows.

 (NCT03720457). Title of the trial-Human CD19 Targeted T Cells Injection (CD19

CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. 25th
October 2018.

 (NCT03720457). Title of the trial-Human CD19 Targeted T Cells Injection (CD19

CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. 25th
October 2018.

 (NCT04003168). Trial name- Human BCMA Targeted T Cells Injection Therapy for
BCMA-positive Relapsed/Refractory Multiple Myeloma. 1st July 2019.

 Prospective identification for the cells in this culture was done by flow cytometry in
which a fluorescent dye Viability Dye and antibodies were used as biological markers
to identify the cells. Statistical methods and JMP software were used to analyse the
result. (Wang et al 2022).

 Results of the study showed that:

Isolation buffers influences the selection of CD3+ cells. DBPS buffer showed better
result compared to XVIVO-15. (Wang et al 2022).

 CD14+ cells do not affect CAR transduction. However, it affects the isolation and
expansion of CD3+ cells.
 incubation didn’t influence CAR-T cell transduction.
 However possible reasons for this were suggested as plastic adhesion of CAR T
cells and poor detection by flowcytometry. Demits of the study include the less
sample size and in efficiency of results.

Merits Limitations
Faster expansion of CAR- T cells due to less sample size and in efficiency of
DPMS media. results.
Repeated with different sample sizes to Results partially supported the aim.
validate the result.
  Study: Allogeneic cell therapy using umbilical cord MSCs on collagen scaffolds for
patients with recurrent uterine adhesion: a phase I clinical trial. (Cao et al 2018)
 Trials:

 (NCT02313415). Trial name: Treatment of Infertility by Collagen Scaffold Loaded

with Umbilical Cord Derived Mesenchymal Stem Cells.10th December 2014

 Prospective identification of this study was done with immunohistochemical analysis

using a microscope and, flowcytometry along with antibodies and Short tandem
repeat (STR) analysis.

 Results showed that

 The intrauterine adhesion score decreased and endometrial thickness in patients
increased. (Cao et al 2018)
 Mesenchymal stem cells from umbilical cord differentiated into patient’s cells. DNA
from the uterine wall was the patients DNA. (Cao et al 2018).

Merits
Screening of patients was done to eliminate
infertility caused by genetics.
Patients of different age groups were
recruited.
Limitations
Symptoms of the patients used in screening
is variable.
Sample size of 26 patients was too small.
Ethical issues due to abortions and
miscarriages.

 Study: Novel lineage depletion preserves autologous blood stem cells for gene
therapy of Fanconi anaemia complementation group A. (Adair et al. 2018).

 Trials:
 (NCT01331018). Trial Name: Gene Therapy for Fanconi Anaemia. Date 7th April
2011.

 Prospective identification of the cells in this study was done using Quantitative real-
time PCR and by flowcytometry.

 Results: (Adair et al. 2018).

 Limited CD34+ cells observed in Fanconi Anaemia patients prior to treatment.

 Lineage depletion T-cell monocyte granulocyte and NK-cell B-cell increased CD34+
transduced cells than the CD34+ cells produced in isolation studies.
 The Transduced cells showed more T-Cell and B-Cell engraftments after
transplantation. (Adair et al. 2018).
 Increased neutrophil and platelet count in patients observed 6 months after
transplantation.
Merits
Modifications to the current study was
made to enrich the CD34+ cells.
Less age groups of patients indicated
that the experiment was suitable for
children
Demerits
Sample size of 3 patients were used in
the experimental trial.
Short term culture of cells.

 Study: Cell therapy induced regeneration of severely atrophied mandibular bone in a

clinical trial. (Gjerde et al. 2018)

 Trials:

 (NCT 02751125). Trial Name: Reconstruction of Jawbone Using Mesenchymal Stem

Cells.

 Prospective identification of the cells was done using flowcytometry and histological
staining. 26th April 2016.

 Results: (Gjerde et al. 2018)

 Successful expansion of mesenchymal stem cells in vitro having the markers CD73,
CD90, and CD105. (Gjerde et al. 2018)
 The scaffolds used in the study degraded and integrated itself into the bone tissue and
aided new bone formation with increased bone volume.
 Cellular interaction was dependent on the bone material.

Merits Limitations
Procedure performed by an experienced Experiment sample size of 13 patients is
surgeon. very small.
The age range of patients in the study was Careful handling of the samples was not
adequate. done as 1 biopsy disintegrated.
Exclusion and inclusion criteria clearly More studies need to be done in vertical
stated. augmentation of the alveolar ridge.

3. How they have helped our understanding of haematopoiesis.

 Haematopoiesis is the mechanism in which blood and plasma cells are formed. It is
controlled by many factors. However, the exact mechanism of haematopoiesis is not
known.
 Most of the evidence came from 2D culture studies rather than 3D studies
 The different culture expansion and proliferation studies of myeloblastic and
lymphoblastic cells suggested that the haematopoiesis is eventful and that specific
subsets of haematopoietic stell cells are influenced by the presence of chemicals
released by other cells and the presence of antibodies and interleukins in the
microenvironments. (Adair et al. 2018).
 Haematopoietic subsets are limited to different lineages and the potency of the cells
decrease as the level of differentiation increases. (Adair et al. 2018).
 Altered Natural killer cells do adapt to the human body microenvironment due to the
cell-to-cell communication through ligands and receptors. (Adair et al. 2018).
 Transplantation of haematopoietic stem cells into different microenvironments such
as bone tissues and muscle tissues promote neovascularisation and differentiation of
progenitors other than blood cells such as osteoblasts. (Gjerde et al. 2018)


4. How they relate to normal physiology, a comparison of 2D vs 3D cells

 2D cultures mostly mimic the immobile cell environments of the body whereas 3D
cultures mimic the mobile cells of the body. ( Kapałczyńska et al. 2018).To culture
conditions like adequate temperature, blood gas concentrations and PH allows the
cells to mimic the behaviour outside the body.

 The physiology of 2D culture is limited as the cells are immobilised on cultures.

( Kapałczyńska et al. 2018). Which is similar to immobilised cells found in the
human body. Only cell to cell interactions is studied in cell culture.

 Cell differentiation, proliferation and cell death is not only dependent on cell-to-cell
interactions but also to cell to environment interactions. Using three dimensional
studies give better evidence of cell-to-cell interaction as well as cell to the
environment interactions are observed. ( Kapałczyńska et al. 2018).

 Two-dimensional cell cultures are maintained on monolayers of agar stating that the
cells are partially adhered to the layer. Sandwich cultures of cells in collagen
replicated the adhesion of cells in the liver that the function and behaviour studied
was more closely related to the body environment. This led to the study of
pharmacokinetics in the liver. (Zeilinger et al 2016).

 2D cultures produce in adequate result when it comes to the clinical trials as the
longevity of cells are reduced either due to cytotoxicity or apoptosis. (Ham et al
2019).
 5. Various types of assays used in haematology their advantages and
limitations.

 Flow cytometry
 Suspensions of cells are fed as a stream of cells into the flow cytometer. This Assay is
used to count cells based on the light scattering principle of cells stained with a
fluorescent dye. As the cells pass through a laser beam, cells are sorted in accordance
with the wavelength of light emitted. (Robinson, 2004).

Advantages
High speed detection of cells depending on
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. (Robinson, 2004).
The equipment used is portable.
Can be available on 3 PMT or 5 PMT.
Disadvantages
Expensive and training needs to be done to
operate the instruments.
Some flowcytometry use two or more lasers
which could lead to errors. (Robinson,
2004).
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. (Robinson,
2004).
Reduction of sensitivity due to the use of
multichannel PMT. (Robinson, 2004).

 Histological staining

 Histological staining involves staining the cells with a dye such as Haematoxylin and
eosin or Masson trichrome to visualise the morphological features to be observed
under the microscope. The sample is deparaffinised and hydrated before staining with
haemoxylin. Then it is counterstained with eosin, dehydrated and mounted for
viewing under the microscope. (Feldman 2004).
Advantages Disadvantages
The morphology of the cells can be Manual work is done.
observed, and the dimensions can be
calculated.
Cost effective and cheap way to calculate Fixatives could degrade cells if used
the number of cells. incorrectly. (Hristu et al 2021).
Cell divisions and activity of the cells can Time consumption of staining the cells.
be studied.
Doesn’t require a large sample size. The slides for histological analysis could be
used once.
Training is not required to study cells under Stains used are specific to the cells.
the microscope.
  RNA sequencing

 RNA is extracted from the cell and isolated before being converted into
complimentary DNA. (Wang et al. 2019). The ends of the sequence is ligated and
sent for amplification by PCR. Then the reads are assembled into transcripts using de
novo assembly and expression levels are analysed. (Corchete et al 2020).

Advantages
Is more accurate in terms of measuring the
quantity and the transcriptomics of the cells
compared to histological staining and
flowcytometry. (Corchete et al 2020).
Offers far more resolution than other
sequencing techniques used today like
microarrays. (Corchete et al 2020).
The processed sample is amplified so more
samples are available for later use.
Sensitive as sequence alignment is detected.
Disadvantages
The use of RNA sequencing should be
handled by a trained professional.
The RNA sequencing requires the use of
complex computers for the analysis of data.
Cost of setting up the equipment and
running costs are high.

The best assay is RNA sequencing as it uses the latest technology and offers a more detailed
and far more accurate quantification of cells.

6. Conclusion

There are a vast majority of cell culture studies done in the field of haematology to find
treatments for immunological diseases like Leukaemia, SCID, multiple myeloma and cancer.
Many studies still need to be conducted to better understand the processes of haematopoiesis,
as the current studies conducted in cell cultures lacks integrity and progression. Cell culture
studies are still at its infancy. However, they are important in understanding the disease
mechanism to and devise a method of drug delivery. The main aim of this critical review was
to critically evaluate cell culture studies done in the field of haematology and provide devise
that 3D cultures offer better in vitro studies than 2D cell cultures. Also, to conclude that RNA
assays provide better results in quantification of cells from culture studies.
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