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LOW TEMERATURE PRESERVATION
Cryopreservation plays an important role in cell and tissue banking. Development of new cryopreservation strategies will attain even greater importance in the future since development of life sciences is a priority direction in Singapore. An efficient cryopreservation method is crucial for reduction experimental variability and transplantation purposes. The NUMI Low Temperature Preservation Unit is an essential service for the Faculty of Medicine, NUS. The purpose of the Unit is to promote research and to improve safety and methodology of low temperature preservation of cells and tissues. Centralisation and professional supervision is necessary to increase quality of cryopreservation procedures. Major directions of the NUMI Low Temperature Preservation Unit activities are research, education and service.
ABOUT CRYOBIOLOGY
Research in the field
of Cryobiology and Cryomedicine includes:
- Cryopreservation
of cells, tissues, and organs
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Theoretical basis
of low temperature preservation · Cryopreservation of tissue
engineered constructs
- Cryoprotective
additives and their pharmacological actions
- Freeze-drying
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Medical applications
of reduced temperatures, cryosurgery, hypothermia +4/28-33C, perfusion
of organs
Results of studies
on low temperature biology and medicine are published in two major journals
- Cryobiology and CryoLetters
Researchers in the
field of cryobiology and cryomedicine are affiliated with two international
societies: Society
for Cryobiology and Society
for Low Temperature Biology
Cryoprotectants
The role of cryoprotectants is to prevent cell/tissue or organ from damage.
Cryoprotectants can de divided by their role in cryopreservation
into two main group
A. penetrating agents
B. non-penetrating agents.
These cryoprotectant agents can be classified by their molecular
weight and other properties:
1. Poly-alcohols - Low Molecular Weight Agents
2. Sugars
3. Polymers - High Molecular Weight Agents
In general, major directions of technology currently under investigations
for cell and tissue preservation can be classified as follows:
1. Hypothermia +4/28-33 °C
1. Hypothermia allows
short term preservation, it could be applied occasionally if method
of cryopreservation is not available or for transportation
purpose
2. Technology based
on freezing concept
2.1. Slow cooling
Slow cooling concept was developed gradually over the past 50 years.
2.1.1.Uncontrolled cooling
2.1.1.1. Using two
subsequent freezers adjusted to -20°C and – 80/86 °C
Initial protocols were developed for non-sensitive types of cells. Briefly,
cells were introduced in a container (e.g. tube) filled with cryopreservation
solutions, and after a short equilibration time they were placed directly
into -20°C freezer for 2 hours and then to –80/86 °C
freezer overnight. This protocol is not suitable for more sensitive
cells, such as the majority of mammalian cells. Cooling in two subsequent
steps was developed at the time a controlled rate freezing device was
not introduced.
2.1.1.2. Cooling in liquid nitrogen vapours
2.1.2 Controlled cooling (using programmed cooling device)
During slow cooling, tissues are usually pre-equilibrated in low
concentration of penetrating cryoprotectant. The concentration of
cryoprotectants is
then gradually increased inside and outside the tissue during several
steps during a 2 hour period. By cooling sufficiently slowly, nearly
all of the available water can be removed from the cells by osmosis.
The advantage of using a programmed cooling device resides in
the possibility to reduce proportion of cells captured by
ice, and to vary the cooling
rate in order to better prepare cells to the temperature of cryostorage.
2.1.2.1 Constant cooling rate
2.1.2.2 More advanced programmes that involve seeding of ice
2.2. Rapid cooling technology
Rapid cooling of biological material involves the direct introduction
of a specimen in solution intermediate concentration (35-40% cryoprotectant)
to the temperature of low temperature storage. Such solute concentration
leads to some of the degree of crystallisation. There is less potential
in pursuing this approach than in “Vitrification technology” described
below since it is more effective to apply solutions which are ice-free
during whole cooling-warming cycle.
3. Vitrification technology
Vitrification is an alternative to customary freezing and this is the
most promising, emerging methodology of cryopreservation. It was generally
thought that supporting solutions for vitrification would be better
for the preservation of living cells and tissues than solutions that
crystallize and hence damage cells during cooling and warming. Vitrification
is of particular importance for medical research because the method
is less time consuming since it does not require the use of specialized
equipment, making it easily applicable in any research laboratories
and clinical settings.
Fig 2. Schematic illustrations of cryopreservation: A. Uncontrolled freezing;
B. Slow cooling procedure that involves seeding of ice; C. Rapid cooling
procedure; D. Vitrification procedure. The colour of the solution reflects
the concentration of solute; the darker the colour the more concentrated
is the solution.
RESEARCH
- Consolidate and accelerate research in the field of low temperature biology and medicine.
- Develop new advanced methods of preservation of living cells and tissues such as vitrification
- Initiate new research collaborative studies on cryopreservation of
- stem cells derived from emerging sources
- blood cells and hematopoietic stem cells
- allografts for cardiovascular surgery
- reproductive cells and tissues
- neuronal stem cells
- tissue engineered constructs and neotissue
- Custom-develop new methods of cryopreservation for clients and research groups.
Current research grants
BMRC grant:
Title:Study on vitrification of cells and tissues
Amount awarded: SGD351,853
PI: Dr. Lilia L. Kuleshova
Co-PIs： A/ Prof. Gavin Stewart Dawe
Defence Science and Technology Agency grant:
Title:Project Freeze
Amount awarded: SGD880,000
PI: Dr. Lilia L. Kuleshova
Co-PI： A/Prof. Lu Jia.
NMRC grant:
Title: The isolation and cryopreservation of male germline stem cells( sper matogonial stem cells) using mouse as a model for the human
Amount awarded: SGD180,588
PI: Dr. Lilia L. Kuleshova
Co-PI： Prof. Paul Watson
Research Team
Dr. Lilia Kuleshova, Ph.D.
Dr. Gouk Sok Siam, Ph.D.
Dr. Wen Feng,Ph.D
Raquel Magalhães, Licenciate
Teo Wan Chun Jotham, B.Sc.
William Shei, MD
Visiting professor: Prof. Allison Hubel
Visiting surgeon: Dr. Wang Xian Wei, MD
HEAD OF UNIT
Dr Lilia Kuleshova
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Dr. Lilia Kuleshova is heading the Low Temperature Preservation Unit at the National University Medical Institutes, National University of Singapore. She began her career in cryobiology in 1982 at the Department of Theoretical Fundamentals of Low Temperature Preservation, Institute for Problems of Cryobiology and Cryomedicine, at the Academy of Sciences of the Ukraine.
She obtained a Ph.D. with specialization in Cryobiology and Cryomedicine in 1992. Since 1995, she continued her work on low temperature biology at the Institute for Reproduction and Development (IRD) at Monash University, Australia. She keeps an Honorary Associate appointment with the University of Sydney. In Australia, she was a principal investigator of three research projects on a new approach in cryopreservation, namely vitrification.
Dr. Kuleshova has significantly contributed to the field of low temperature biology during her 25 years of international career in cryobiology and cryomedicine. |
Her research interests include Fundamentals of Low Temperature Preservation and lately are more focused on clinical application. She has consistently published results on development of diagrams of phase and physical transitions of cryoprotectants, cryomicroscopy and freeze-drying; vitrification of human oocytes, embryos, neuronal stem cells and mesenchymal stem cells -matrix/scaffold systems. Dr. Kuleshova was first who established a procedure which eliminates risk of contamination in cryopreservation of stem cells and embryos. Her research brings prospects for cryopreservation of more complex living objects.
In the field of reproductive medicine, Dr. Kuleshova's research on vitrification of human oocytes and embryos is recognized worldwide. Dr. Kuleshova was the first scientist to achieve vitrification of human oocytes and later this research has produced the first live birth. Her research was focused on cryopreservation of human embryos and oocytes at IRD (Monash University), and Medical Research Centres in Tokyo and Kobe. She has received invitations from two major Medical Universities in Japan (Jikei University and Keio University) where she consequently presented lectures. She was invited to give a talk to several hundred medical professionals from all over Japan in Labour Square Hall in Tokyo. In Japan, she was interviewed by major newspapers and she was reported in two articles in Mainichi Shimbun, one of the largest newspapers in Japan with distribution of approximately four million copies a day. Dr. Kuleshova has also carried out her research as an invited scientist at Zurich University in Switzerland and in Bologna, Italy.
Dr Kuleshova has joined the National University of Singapore (NUS) in 2003. Since then she has established and lead the Low Temperature Preservation Unit (LTPU) at the National University Medical Institutes (NUMI) at Yong Loo Lin School of Medicine, NUS. She is a member of Cardiovascular Themed Group and closely collaborates with the Heart Institute, NUH. Since establishing LTPU, specialised advisory service on cryobiology and cryomedicine is continuously provided to the staff of Departments of Yong Loo Lin School of Medicine, Departments of NUS, NUH and General hospital. Additionally, lectures on Cryobiology and Cryomedicine were given at a number of departments of NUS, NUH and at HSA. A course on "Preservation of Cells, Gametes, Native and Engineered Tissues" which presents an overview of the scientific foundation of cryobiology and cryomedicine was designed by Dr. Lilia Kuleshova. LTPU conducts this course in the form of a specially designed workshop with practical classes. Visiting professors in the field of cryobiology also invited, giving the course a broader and international scope. Dr. Kuleshova also was invited to present lectures at Leibnitz University (Germany), Monash University (Australia), Tokyo Women’s University (Japan), and the University of London (UK).
In 2007, as recognition of the important contribution which Dr. Kuleshova has made to the fields of Cryobiology and Cryomedicine, the Society for Low Temperature Biology has elected her for Committee Member, which involves renowned scientists from all across the world. She is referee of 6 international journals (Cryobiology, CryoLetters, Reproduction Fertility and Development, Human Reproduction, Tissue Engineering, Biomaterials)
At NUS, Dr. Kuleshova has been actively engaged in Cryopreservation of tissue engineered constructs, stem-cells, red blood cells and the development of “safe” technologies including protein/serum-free cryopreservation solutions. With the support of a successful competitive grant by the Biomedical Research Council (BMRC), Dr. Kuleshova’s research has a potential impact in
1. Benefiting patients requiring urgent metabolic support during the time of liver failure by developing cryopreservation of systems involving hepatocytes;
2. Plays an important role in establishing the foundation for successful cryopreservation of bone marrow mesenchymal stem cell based tissue engineered constructs for bone and cartilage regeneration and creating a platform for off the shelf tissue engineered constructs
3. neuronal stem cell based brain regeneration research
Dr Kuleshova is also a principal investigator of project Freeze which is an interinstitutional collaboration. This high priority research project is commissioned and funded by DSTA. The expected outcomes of these investigations conducted at the Yong Loo Lin School of Medicine, will allow direct transfusion of thawed red blood cells to patients in emergency situation. The results will also benefit the Centre for Transfusion Medicine.
Dr. Kuleshova is currently engaged in the study of cryopreservation by vitrification of human mesenchymal stem cells in alginate beads culture system and more complex system involving scaffolds. She aims to contribute in the field by investigating whether phenotype induction has an impact on the outcomes of cryopreservation. In forthcoming years, development of efficient cryopreservation of stem cells derived from a variety of sources will be also part of her integral efforts which is of great importance given the current standing of Singapore government in Medical Research. |
RECENT PUBLICATIONS
1. Kuleshova, L., Gianoroli, L., Magli, C., Ferraretti, A., Trounson, A. (1999) Birth following vitrification of small number of human oocytes. Hum. Rep. 14(12): 3077-3079.
2. Kuleshova, L. L., Shaw J. M. (2000). A strategy for rapid cooling of embryos within a double straw to eliminate the risk of contamination during cryopreservation and storage. Hum. Rep.15, 2604-2609.
3. Kuleshova, L.L., Shaw, J.M., Trounson, A.O. (2001). Studies on replacing most of the
penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology. 43(1):21-31.
4. Kuleshova, L.L., Lopata, A. (2002). Vitrification can be more favorable than slow cooling.
Fertil. Steril. 78(3):449-454.
5. Kuleshova L., Wang, X.W, Y. Wu, Y., Zhoi, Yu. H. (2004) Vitrification of encapsulated
Hepatocytes with reduced cooling/warming rates. CryoLetters 25 (4): 241-254
6. Kuleshova, L. L., Gouk, S. S. and Hutmacher, D.W. (2007) Vitrification as a prospect for cryopreservation of tissue-engineered constructs. Biomaterials, 28:1585-1596
7.Yingnan Wu, Hanry Yu, Shi Chang, Raquel Magalhaes, and L.L. Kuleshova (2007) Vitreous cryopreservation of cell-biomaterial constructs involving encapsulated hepatocytes, Tissue Engineering, 13: 649-658
8.Tan, C. K. F., Lee, K. H., Gouk, S. S., Magalhães, R., Poonepalli, A., Hande, M. P., Dawe, G. S., Kuleshova, L. L. Optimization of cryopreservation of stem cells cultured as neurospheres: comparison between vitrification and slow-cooling and rapid cooling “freezing” protocols. Cryo Letters 28, 445 (2007)
9 Kuleshova, L. L., and D. W. Hutmacher (2008) Cryobiology, in C. van Blitterswijk, ed., Tissue Engineering: Biomedical Engineering: London, Elsevier, p. 363-401
10.Magalhães, R., Wang, X.W., Gouk, S. S., Lee, K.H., Ten, C.M.,Yu H and Kuleshova L. Vitrification successfully preserves hepatocytes spheroids. Cell Transplantation 17, in press, (2008)
11.Kuleshova, L. L., Tan, C. K. F., Magalhães, R., Gouk, S. S., Lee, K. H., Dawe, G. S. Effective cryopreservation of neuronal stem cells or progenitor cells without serum or proteins by vitrifcation. Cell Transplantation, accepted( 5th May, 2008).
Click picture for larger view.
RESEARCH PROJECTS PROPOSED FOR MASTER AND PHD STUDENTS
Undergraduate students are also welcome!
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Study on preservation of blood cells by vitrification
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Vitrification of bone marrow derived mesenchymal stem cells involving alginate-fibrin beads
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Evaluation of the proliferation and multipotent differentiation of neuronal stem cells preserved by serum and protein-free vitrification
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Study on vitrification of vascular grafts and heart valves
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Formation, culture, and vitreous preservation of cell spheroids
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Proliferation and differentiation of bone marrow derived mesenchymal stem cells cultured on scaffolds after vitrification
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Study on vitrification of cancer cell lines and T-cells
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EDUCATION
Dr. Kuleshova, Head, LTPU, has prepared and introduced lectures on Cryobiology and Cryomedicine for postgraduate/undergraduate students at NUS. As the member of committee of international Society for Low temperature Biology, she was the first to introduce this area of study in Singapore.
Establish courses on advances in cryobiology, hypothermic preservation and freeze-drying, and practical tutorial for scientific community, staff of commercial companies and students. |
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A course which presents an overview of the scientific foundation of cryobiology and cryomedicine was designed by Dr. Lilia Kuleshova. The course was conducted in the form of a specially designed workshop with practical classes. Visiting professors in the field of cryobiology were invited, giving the course a broader and international scope.
Provides on-going training for clients on cryopreservation methods with emphasis on vitrification procedures, which are achieved by direct immersion into liquid nitrogen of pre-treated living cells/tissues.
Provides advice for clients on the best current practice and update them with knowledge of modern methods.
Click picture for larger view.
SERVICE
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-Assist
in application of programmed cooling device. Please contact our staff
(phone 6516 3767) for using programmed cooling machine located
at LTPU.
-Improvement
of cryopreservation protocols and methodology of pre- and post-care
currently
used in clients' practice.
-Work
with clients to co-develop new protocols on cryopreservation of
living cells and
tissues.
-Gradual
transition of samples' storage in liquid nitrogen to cryostorage
at optimal temperatures
in liquid nitrogen vapors.
-Storage
of valuable samples (back up for clients).
-Ensure
the safe cryopreservation of tissues and cells by
- preventing likelihood of spreading viruses among preserved specimens
and from specimens to staff by implementing regulations recommended
by the Society for Cryobiology and the Society for Low Temperature
Biology, and
- application
of animal/human origin-free solutions for cryopreservation
-Provide kits of
solutions for vitrification and tools for safe cryopreservation.
-Assist in application
of cryosurgical instruments in medicine.
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CONTACT
Low Temperature Preservation
Unit
National University Medical Institutes
Faculty of Medicine
National University of Singapore
Office: #03-01C, Lab: #03-18
Block MD11,
10 Medical Drive
Singapore 117597
Fax: 6773 5461
Tel: 6516 -3359 (office) -3767(lab)
E-mail:nmikl@nus.edu.sg
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