BIOL5368: Designing a Research Project Stem Cells

Clinical-Scale Cellular
Production for Nanokicking
Technology through the use of
Hollow Fibre Bioreactors

I GUSTI NGURAH KRISHNA PRIYAKA (2586619)

Supervisor:
Prof. Matt Dalby & Mark Sprott, PhD

TABLE OF CONTENTS
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I. Outline of research project……………………………………………………………………………….. 1

II. Lay language summary…………………………………………………………………………………….. 1
III. Aims……………………………………………………………………………………………………………….. 2
IV. Hypothesis……………………………………………………………………………………………………… 2
V. Research plan………………………………………………………………………………………………….. 2
VI. Ethical consideration………………………………………………………………………………………. 6
VII. Strategy of dissemination………………………………………………………………………………. 6
VIII. Potential impacts………………………………………………………………………………………….. 6
References…………………………………………………………………………………………………………… 8
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I.OUTLINE OF RESEARCH PROJECT

Nanokicking technology has emerged as a novel strategy to induce osteogenesis
differentiation of human mesenchymal stem cells (hMSCs). By mechanically stimulating
hMSCs with 1 kHz vibrations within nanometre amplitudes, we can provide cells with the
necessary mechanotransductive signals for osteogenesis. Due to its chemical-free
stimulation, this approach might show many benefits in clinical applications (Nikukar et al.,
2013; Campsie et al., 2019). High cell yield, sufficient for clinical dosage, is required to bring
the advantages of nanokicking to patients. Furthermore, regulations necessitate cell
production methods to comply with Good Manufacturing Practice (GMP) to ensure their
safety. (Nold et al., 2013; Campsie et al., 2019). Hollow fibre bioreactors (HFB), which are
closed cell culture systems offer potential solutions for these requirements by providing
high yields and more reproducible results (Nold et al., 2013; Frank et al., 2019). This
research project will investigate the upscaling effort of manufacturing clinical-scale hMSCs
for nanokicking application through the use of HFB. A literature review will be undertaken,
in which current issues of expanding cell production for nanokicking and the ideal
requirements will be identified. The results of this review, as well as data from relevant
biological assays will then be used to formulate recommendations regarding the ideal
method in upscaling cell production for nanokicking.

II. LAY LANGUANGE SUMMARY

Stem cells has gained a lot of popularity in recent years due to their ability to
regenerate into different types of cells, including bone cells. Until recently, in order to turn
stem cells into bone-forming cells, approaches which involve exposing stem cells to various
chemical substances have been explored. The use of chemical substances for stem cells
however have been known to be cumbersome and produce inconsistent results, in addition
to their associated side effects (James et al., 2016; Campsie et al., 2019; Orapiriyakul et al.,
2020). Alternatively, stem cells have been shown to respond to various physical
stimulations, including small vibration forces or nanovibrations. By solely subjecting stem
cells to nanovibrations, we can also control their fate and turn them into bone cells. This
strategy offers potential benefits for clinical use by providing a new source for bone grafts,
without side effects often associated with using chemical substances (Nikukar et al., 2013;
Campsie et al., 2019; Orapiriyakul et al., 2020). In order to bring these benefits to patients, a
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safe and efficient method must be developed to produce a large number of stem cells.
Hollow fibre bioreactors, which are closed, automated systems used to grow a large number
of cells, may offer potential solutions. To this end, this research project will try investigate
the current challenges in upscaling the cell productions for nanovibrations technology and
the ideal cell production method in order to translate nanovibrations technology to clinical
settings.

III. AIMS
The aims of this project include:
 To gain insights into the clinical translation of nanokicking technology within its role
to stimulate osteogenesis and different methods of ex vivo cellular expansion that
may support its clinical translation.
 To identify issues in ex vivo cell expansion for nanokicking, specifically looking at the
gap between the current state of the cell production strategy and the ideal
requirements within the context of GMP.
 To connect the dots between the theoretical and practical aspect of potential GMP-
compliant methods and protocols in upscaling cell production for nanokicking.
 To formulate recommendations for future studies.

IV. HYPOTHESIS
Hollow fibre bioreactors cell culture is an ideal method of clinical-grade hMSCs
expansion for nanokicking in compliance with Good Manufacturing Practice.

V. RESEARCH PLAN
Initially, this research project will take the form of a systematic literature review, where
a number of relevant results from various literature will be recapitulated in a narrative
manner. The complete report of this project will be written as a master's dissertation and
will consist of the following sections: introductions, literature review, methodology, results,
discussion, and conclusion. The scope of the study should be defined first to answer its
hypothesis in a concise and effective manner. In this research, these primary questions act
as the limits which will help to focus the scope of this study:
 What are the approaches which have been explored in cell manufacturing for
nanokicking?
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 Is HFB cell culture an ideal method for clinical-scale cell manufacturing in

nanokicking?
Since this project will be carried out under a time constraint, it is also essential to
organise the research timeline efficiently. This study will start on 12 th of July 2021 and will be
divided into several stages: 
1. Literature searching 
2. Literature review
3. Result analysis
4. Revision and dissemination preparation
Initially, in the literature searching stage, efforts will be focused on finding relevant
literature on clinical translation of hMSCs-based therapy, upscaling hMSCs production,
nanokicking, and HFB. Searching of literature will then be performed by utilising online
libraries, such as Google Scholar or PubMed. Several keywords, for example, “upscaling MSC
cell production”, “hollow fibre bioreactors for MSC production”, “nanovibrations MSC
stimulation”, will be applied in order to retrieve specific search results. The literature in the
search results are then sorted according to its relevancy. The literature searching stage will
take place in the first 2 weeks of the project, where the writing of the dissertation's
introduction section will also be performed. The general structure of the project and
dissertation may also be re-evaluated during this stage. These structures should be
coherent, and concisely retain relevant information.
In the literature synthesis stage, a critical appraisal of each included literature will be
performed first to assess their methodology, specifically looking at the design of the studies
and the quality at which they were conducted. Afterwards, any relevant information from
these literature will be extracted and put within the context of this project. A table then can
be utilised to show and highlight the results of the assessed studies. This table will include
essential information regarding the studies, such as the studies' title, authors, summaries of
methodologies, statistical aspects, strengths/weaknesses and the implications of the
studies' findings. This activity intends to give a “bird's-eye view” of the current landscape in
the clinical translation of hMSCs-based therapy and HFBs in general. To put this information
into the project's perspective, a list of ideal criteria for methods of upscaling cell production
will be formulated. These criteria will then be used to evaluate the feasibility of HFBs for
nanokicking cell expansion by looking at some variables, such as cell yield, nanokicking
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applicability, GMP compliance, cost and time, while also bearing in mind its practical aspect.
This literature synthesis stage will be carried out in the second month of the project and is
estimated to take one month to finish.
In the following result analysis stage, the criteria mentioned above will be utilised to
identify the current issues and challenges in upscaling cell production for nanokicking by
using hollow fibre bioreactors, as well as the potential advantages. Unpublished data from a
number of cell assays will also be included to support the findings of literature review. These
assays will include reverse transcription quantitative polymerase chain reaction (RT-qPCR)
for osteogenic markers to look at the osteogenesis process. Results from the static control
group, which are hMSCs cultured in HFB without nanokicking, as well as results from hMSCs
cultured with flat flasks, will be compared with results from the HFB-cultured nanokicking
group under identical timescale. This comparison will then be statistically tested for
significance, with p value below 0.05 considered as significance. Next, these topics are going
to be discussed elaborately in the discussion section of the dissertation, describing the
implications and significances of this project's findings. The discussion section will also touch
on some of this study's own possible limitations, which might come from risks of bias, which
are systematic errors that may come from the inclusion/exclusion criteria of studies that will
be chosen, methods of synthesising and extracting information from the studies, and the
general methodology of data analysis in this project.
Recommendations for future studies based on this research's findings will be made
and included in the conclusion section of the dissertation. These recommendations are
inferred from the results of this study, covering a few aspects which hopefully bridge further
the theoretical and practical aspects in the future of upscaling cell production for
nanokicking. Finally, after the entire project, as well as the writing of the dissertation is
completed, the dissertation will be revised for further improvements, and the dissemination
preparation will start in the last stage. The dissemination for this project is discussed in the
following "Strategy for Dissemination" section of this report. The complete research
timeline is expected to be four months long, extending from July to November 2021. The
Gantt chart below summarises the research timeline. 

Figure 1. Gantt chart summarising the timeline of this research project.

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July 2021 August 2021 September October 2021 November

2021 2021
Tasks
Week Week Week Week Week Week Week Week Week Week
1-2 2-4 1-2 2-4 1-2 2-4 1-2 2-4 1-2 2-4
th st
1. Literature searching (July 12 -31 2021)
Literature
searching
Writing the
introductory
section of the
dissertation
and re-
evaluation of
project
structure
2. Literature review (July 26th-August 23rd 2021)
Literature
review on
nanokicking
and its clinical
translation
Literature
review on HFB
3. Result analysis (August 16th – September 6th 2021)
Processing
result data
Writing the
result,
discussion and
conclusion
section of
dissertation
4. Revisions and dissemination preparation (September 6th-September 27th-October 11th 2021)
Revisions of
the
dissertation
write up
Preparation for
dissemination

VI. ETHICAL CONSIDERATION

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There are no ethical issues which are directly related to this research project. The
use of hMSCs does not raise any ethical concerns, since they can be derived from adipose
tissues umbilical cord blood, and bone marrow, in contrast to embryonic stem cells
(Volarevic et al., 2018).

VII. STRATEGY FOR DISSEMINATION

The main purpose of dissemination is to ensure that the benefits of the research is
translated properly to the wider community in academia or beyond. In formulating a
dissemination strategy, there are a number of aspects to consider, such as the specific aims,
the target audiences and the means for the dissemination (NIHR Dissemination Centre,
2017; Purssell and McCrae, 2020). The aim of dissemination for this research is to familiarise
the target audiences with the potential methods of upscaling cell production in an effort to
translate nanokicking technology into clinical settings. The target audiences for this research
dissemination will include academia researchers as the main audience, clinicians, various
stake holders/decision makers, and the general public. Researchers are regarded as the
main target audience because they will most likely to receive the potential benefits from
this research directly. As for the means of dissemination for this research, they will need to
be designated specifically for each target audiences to ensure effective and efficient
communications. For example, journal publications and conferences are more appropriate
for target audiences such as researchers or clinicians, whereas online articles and social
media are more ideal for general public.

VIII. POTENTIAL IMPACTS

Since this research project concerns with the clinical translation of nanokicking, the
potential impacts of this project lie on how nanokicking might be used in a hMSC-based
treatment as bone grafts. There are sizeable demands for bone grafts, as bone is one of the
most transplanted tissues in humans. However, at the present time, these demands are
inadequately met by autograft and allograft bone donors alone. Moreover, these sources of
bone grafts are associated with many issues, concerning their low availability, viability, and
efficacy (James et al., 2016; Orapiriyakul et al., 2020). Nanokicking has been shown to
physically stimulate hMSCs to differentiate into osteoblasts. Therefore, tissue engineering
approaches using nanokicked hMSCs might present a novel source for bone grafts.
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Additionally, this approach does not involve the use of biochemicals, which are often
associated with several side effects (James et al., 2016; Campsie et al., 2019; Orapiriyakul et
al., 2020). In order to bring these benefits into clinical settings, an ideal method of cell
production is required, which this research project will seek to investigate.

REFERENCES:
Campsie, P. et al. (2019) ‘Design, construction and characterisation of a novel
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nanovibrational bioreactor and cultureware for osteogenesis’, Scientific Reports,

9(1), pp. 1–12. doi: 10.1038/s41598-019-49422-4.

Frank, N. D. et al. (2019) ‘Evaluation of reagents used to coat the hollow-fiber bioreactor
membrane of the Quantum® Cell Expansion System for the culture of human
mesenchymal stem cells’, Materials Science and Engineering C, 96(September 2018),
pp. 77–85. doi: 10.1016/j.msec.2018.10.081.

James, A. W. et al. (2016) ‘A Review of the Clinical Side Effects of Bone Morphogenetic
Protein-2’, Tissue Engineering - Part B: Reviews, 22(4), pp. 284–297. doi:
10.1089/ten.teb.2015.0357.

NIHR Dissemination Centre (2017) ‘Getting your message heard - and used How to
disseminate your research : Your dissemination plan : things to consider’, National
Institute for Health Research. Available at: https://www.nihr.ac.uk/funding-and-
support/documents/funding-for-research-studies/manage-my-study/How-to-
disseminate-your-research/dissemination-guidance.pdf%0Ahttps://www.arc-
swp.nihr.ac.uk/uploads/attachments/NIHR Dissemination Guidance.pdf.

Nikukar, H. et al. (2013) ‘Osteogenesis of mesenchymal stem cells by nanoscale

mechanotransduction’, ACS Nano, 7(3), pp. 2758–2767. doi: 10.1021/nn400202j.

Nold, P. et al. (2013) ‘Good manufacturing practice-compliant animal-free expansion of

human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-
based bioreactor’, Biochemical and Biophysical Research Communications, 430(1),
pp. 325–330. doi: 10.1016/j.bbrc.2012.11.001.

Orapiriyakul, W. et al. (2020) ‘Nanovibrational Stimulation of Mesenchymal Stem Cells

Induces Therapeutic Reactive Oxygen Species and Inflammation for Three-
Dimensional Bone Tissue Engineering’, ACS Nano, 14(8), pp. 10027–10044. doi:
10.1021/acsnano.0c03130.

Purssell, E. and McCrae, N. (2020) ‘How to Perform a Systematic Literature Review’, How to
Perform a Systematic Literature Review. doi: 10.1007/978-3-030-49672-2.

Volarevic, V. et al. (2018) ‘Ethical and safety issues of stem cell-based therapy’, International
Journal of Medical Sciences, 15(1), pp. 36–45. doi: 10.7150/ijms.21666.