Cytotherapy, 2014; 0: 1e3
Cryopreservation can be used as an anti-aging strategy
MAHMOOD S. CHOUDHERY1,2 & DAVID T. HARRIS2 1
Tissue Engineering and Regenerative Medicine Laboratory, Advance Center of Research in Biomedical Sciences/King Edward Medical University/Mayo Hospital, Lahore, Pakistan, and 2Department of Immunobiology, College of Medicine, the University of Arizona, Tucson, Arizona, USA
Key Words: adipose tissue, anti-aging, cryopreservation, mesenchymal stem cells
Advanced age is a major risk factor for various diseases and disorders (such as cardiovascular and neurodegenerative disorders), highlighted by diminished repair and differentiation potential of stem cells limiting their potency for treatment. Recent achievements in the field of tissue engineering and regenerative medicine are very promising. The preclinical data that used stem cells isolated from adult tissues such as adipose, bone marrow and neonatal sources (umbilical cord blood and cord tissue) are very auspicious. As a result, several hundred preclinical and clinical trials have been registered with the US Food and Drug Administration that are at various stages of completion (see www.clinicaltrials. gov). Although these technological advancements have opened new possibilities for the treatment of various diseases, there are a number of issues still to resolve. For example, neonatal sources are rich in stem cells but can be obtained only once in a lifetime. Autologous stem cell therapy appears the most practical option for cell-based therapy; however, the regenerative ability of stem and progenitor cells is severely compromised with increasing donor age. Similarly, autologous stem cells such as those obtained from adipose tissue or bone marrow can be used for cell therapy or for tissue engineering only if available in sufficient number. Although expansion may provide increased cell numbers and higher purity, it leads to reduced cell function (limits proliferative and differentiation capabilities) as the result of cell aging, increased pecuniary cost and increased risk of contamination with microorganisms. Therefore, preservation of stem and progenitor cells (from adipose tissue and bone marrow) at a young age (or at the time of delivery in the case of cord blood and
cord tissue) while maintaining their youthful biological activities and potential could provide an ideal situation for future regenerative medicine applications. In addition, cryopreservation of cells and tissues could avoid repeated in vitro culture expansion because the cells and tissues obtained during different procedures at various time points could be cryopreserved and pooled at the time of care when specific medical conditions need transplantations. Many strategies have recently been used to address the age-related functional impairment of stem cells. These approaches include treatment with cyclin-dependent kinase inhibitors [1], exposure to low temperatures [2] and over-expression of antioxidant genes such as sirtuins and heat-shock proteins [3]. Unfortunately, the methods that increase age-related dysfunction of stem cells can also increase chances of other diseases. For example, cyclin-dependent kinase inhibitors such as p16INK4a may increase the chance of cancer development. Other approaches such as culturing of cells at low temperatures are more effective for young cells rather than aged cells [3]. Therefore, other anti-aging strategies are required that are safe and effective and that do not compromise biological properties of cells. Recent studies have demonstrated no negative effect of cryopreservation on the functionality of stem and progenitor cells obtained from various sources [4,5]. Harvesting tissues (such as adipose and bone marrow tissues) at the time of treatment may put the donor at risk. However, if cells and tissues collected from young donors can be cryopreserved and still maintain their biological activities, it would be ideal for future applications in tissue engineering and regenerative medicine. Therefore, we hypothesize
Correspondence: Mahmood S. Choudhery, PhD, Tissue Engineering and Regenerative Medicine Laboratory, Center of Research in Biomedical Sciences, King Edward Medical University/Mayo Hospital, Lahore, Pakistan. E-mail: [email protected] (Received 3 July 2014; accepted 23 August 2014) http://dx.doi.org/10.1016/j.jcyt.2014.08.012 ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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M. S. Choudhery & D. T. Harris
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D
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Figure 1. (A) Flow cytometric analysis of cells (n ¼ 3 each) indicate that both fresh and frozen-thawed adipose tissueederived MSCs had positive expression of CD44, CD73, CD90 and CD105 and negative expression of hematopoietic markers CD3, CD14, CD19, CD34 and CD45. (B) Growth characteristics of fresh and cryopreserved MSCs were compared (n ¼ 8 each). Both fresh and cryopreserved MSCs showed equivalent number of population doublings. Similarly, doubling time was also comparable in both fresh and cryopreserved MSCs. Colony-forming unit assay on day 14 indicated equivalent percentage plating efficiency [(No. of colonies counted/No. of cells plated) 100] for MSCs harvested from fresh or cryopreserved adipose tissue. (C) Adipogenic induction in fresh and cryopreserved adipose tissueederived MSC cultures (n ¼ 5) was assessed by treating MSCs (fresh and cryopreserved) with adipogenic induction medium for 21days. Differentiation into adipocytes was compared by means of histochemistry, immunofluorescence and RT-PCR. Equivalent number of oil red Oepositive cells and oil red O uptake along with immunofluorescence staining for FABP4 fatty acid binding protein 4 (FABP4) and mRNA levels of lipoprotein lipase (LPL) and peroxisome proliferator-activated-receptor-gamma (PPAR-g) between fresh and cryopreserved adipose tissue-MSCs revealed no significant difference. (D) MSCs from both sources (n ¼ 5) show similar osteogenic induction as detected by von Kossa stain of matrix mineralization deposition (analyzed with the use of Image J software), immunofluorescence staining for osteonectin and quantitative analysis of osteogenic-associated gene expression (osteocalcin and alkaline phosphatase) with RT-PCR. (E) Chondrogenic potential (n ¼ 5) of fresh and cryopreserved MSC cultures was also similar, as indicated by quantification of alcian blue staining and quantitative RT-PCR for mRNA expression of aggrecan and collagen type 2. (F) Number of neuron-like cells, nestin expression as determined by immunofluorescence staining and RT-PCR for the expression of neurogenic specific genes (NSE [neuron-specific-enolase], NFM [neurofilament]) indicated equivalent neurogenic potential in fresh and cryopreserved MSC cultures (n ¼ 5).
that cryopreservation of such cells and tissues (being an experimental intervention) can safely and reproducibly maintain the youthful functional capabilities of the tissues and in turn the cells contained within and therefore can be used as an anti-aging strategy. The processes of cryopreservation and thawing as well as concentration and type of cryoprotectant play an important role in maintaining overall biological characteristics of cells. Therefore, different methods of both cryopreservation and thawing (most commonly slow- and rapid-cooling methods) and type and concentration of cryoprotectant (phosphate buffer saline human albumin, dimethyl sulfoxide, ethylene glycol and sucrose) have been used to get
the desired outcomes such as viability, proliferation and differentiation potential. Although previous reports in the literature are varied, the methodology that we used can successfully cryopreserve cells/tissues in a liquid nitrogen container and later thaw the cells/tissues for clinical use [4,5]. The experiments performed in our laboratory in this regard are very promising (Figure 1). We have collected adipose tissue from donors of various ages, which were processed, cryopreserved and stored frozen in liquid nitrogen for extended periods of time. On thawing, the samples were evaluated for cellular content and viability (in particular, mesenchymal stem cell [MSC] content) as well as for potential utility for
Cryopreservation as an anti-aging strategy regenerative medicine and/or tissue engineering applications. Potential clinical utility was evaluated by use of growth characteristics as well as the capacity to differentiate into mesenchymal and non-mesenchymal lineages. The results indicate that cryopreserved cells maintain morphology and high viability. Fluorescence-activated cell sorting analysis confirmed that cells isolated from both fresh and frozen tissues expressed a similar percentage of cells with MSC markers. Long-term in vitro expansion of fresh and cryopreserved cells indicated no difference in either the number or time of population doublings. In addition, it was observed that cryopreservation does not have a significant effects on senescent features (evaluated at passages 1, 5 and 8) of MSCs. MSCs isolated from fresh and frozen tissue were capable of differentiating along adipogenic, chondrogenic, osteogenic and neurogenic pathways, as confirmed by histology and real-time polymerase chain reaction (RT-PCR) analysis of tissue-specific messenger RNAs (mRNAs). No significant functional differences were observed between MSCs from frozen tissue as compared with fresh tissue [4,5]. Similar experiments have also been performed by preserving isolated cells rather than whole tissues. Overall, these findings indicate that storing the cells of healthy and young donors at ultralow temperatures can maintain their youthful properties. The purpose of this study was to show that longterm cryopreservation does not affect the biological properties of whole tissues or cells contained in it. Stem cell banks offer the opportunity to preserve
3
stem cells in viable conditions for prolonged periods of time until needed by the donor. The patients undergoing liposuction or other cosmetic procedure thus have the chance to preserve some tissue/cells when healthy and young that could be used later for tissue engineering and regenerative medicine applications. Because donor age negatively regulates stem cells, storing the cells at low temperatures can maintain their youthful properties, and therefore the strategy can be used as an anti-aging strategy.
Disclosure of interests: Dr Choudhery does not have financial or non-financial competing interests. Dr Harris is the CSO for Adicyte, Inc. References [1] Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, et al. Stem-cell ageing modified by the cyclin dependent kinase inhibitor p16INK4a. Nature 2006;443: 421e6. [2] Stolzing A, Sethe S, Scutt AM. Stressed stem cells: temperature response in aged mesenchymal stem cells. Stem Cells Dev 2006;15:478e87. [3] Rattan SI, Singh R. Progress and prospects: gene therapy in aging. Gene Ther 2009;16:3e9. [4] Choudhery MS, Badowski M, Muise A, Harris DT. Utility of cryopreserved umbilical cord tissue for regenerative medicine. Curr Stem Cell Res Ther 2013;8:370e80. [5] Choudhery MS, Badowski M, Muise A, Pierce J, David DT. Cryopreservation of whole adipose tissue for future use in regenerative medicine. J Surgical Res 2014;187:24e35.
Cryopreservation can be used as an anti-aging strategy
MAHMOOD S. CHOUDHERY1,2 & DAVID T. HARRIS2 1
Tissue Engineering and Regenerative Medicine Laboratory, Advance Center of Research in Biomedical Sciences/King Edward Medical University/Mayo Hospital, Lahore, Pakistan, and 2Department of Immunobiology, College of Medicine, the University of Arizona, Tucson, Arizona, USA
Key Words: adipose tissue, anti-aging, cryopreservation, mesenchymal stem cells
Advanced age is a major risk factor for various diseases and disorders (such as cardiovascular and neurodegenerative disorders), highlighted by diminished repair and differentiation potential of stem cells limiting their potency for treatment. Recent achievements in the field of tissue engineering and regenerative medicine are very promising. The preclinical data that used stem cells isolated from adult tissues such as adipose, bone marrow and neonatal sources (umbilical cord blood and cord tissue) are very auspicious. As a result, several hundred preclinical and clinical trials have been registered with the US Food and Drug Administration that are at various stages of completion (see www.clinicaltrials. gov). Although these technological advancements have opened new possibilities for the treatment of various diseases, there are a number of issues still to resolve. For example, neonatal sources are rich in stem cells but can be obtained only once in a lifetime. Autologous stem cell therapy appears the most practical option for cell-based therapy; however, the regenerative ability of stem and progenitor cells is severely compromised with increasing donor age. Similarly, autologous stem cells such as those obtained from adipose tissue or bone marrow can be used for cell therapy or for tissue engineering only if available in sufficient number. Although expansion may provide increased cell numbers and higher purity, it leads to reduced cell function (limits proliferative and differentiation capabilities) as the result of cell aging, increased pecuniary cost and increased risk of contamination with microorganisms. Therefore, preservation of stem and progenitor cells (from adipose tissue and bone marrow) at a young age (or at the time of delivery in the case of cord blood and
cord tissue) while maintaining their youthful biological activities and potential could provide an ideal situation for future regenerative medicine applications. In addition, cryopreservation of cells and tissues could avoid repeated in vitro culture expansion because the cells and tissues obtained during different procedures at various time points could be cryopreserved and pooled at the time of care when specific medical conditions need transplantations. Many strategies have recently been used to address the age-related functional impairment of stem cells. These approaches include treatment with cyclin-dependent kinase inhibitors [1], exposure to low temperatures [2] and over-expression of antioxidant genes such as sirtuins and heat-shock proteins [3]. Unfortunately, the methods that increase age-related dysfunction of stem cells can also increase chances of other diseases. For example, cyclin-dependent kinase inhibitors such as p16INK4a may increase the chance of cancer development. Other approaches such as culturing of cells at low temperatures are more effective for young cells rather than aged cells [3]. Therefore, other anti-aging strategies are required that are safe and effective and that do not compromise biological properties of cells. Recent studies have demonstrated no negative effect of cryopreservation on the functionality of stem and progenitor cells obtained from various sources [4,5]. Harvesting tissues (such as adipose and bone marrow tissues) at the time of treatment may put the donor at risk. However, if cells and tissues collected from young donors can be cryopreserved and still maintain their biological activities, it would be ideal for future applications in tissue engineering and regenerative medicine. Therefore, we hypothesize
Correspondence: Mahmood S. Choudhery, PhD, Tissue Engineering and Regenerative Medicine Laboratory, Center of Research in Biomedical Sciences, King Edward Medical University/Mayo Hospital, Lahore, Pakistan. E-mail: [email protected] (Received 3 July 2014; accepted 23 August 2014) http://dx.doi.org/10.1016/j.jcyt.2014.08.012 ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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M. S. Choudhery & D. T. Harris
A B
C
D
E
F
Figure 1. (A) Flow cytometric analysis of cells (n ¼ 3 each) indicate that both fresh and frozen-thawed adipose tissueederived MSCs had positive expression of CD44, CD73, CD90 and CD105 and negative expression of hematopoietic markers CD3, CD14, CD19, CD34 and CD45. (B) Growth characteristics of fresh and cryopreserved MSCs were compared (n ¼ 8 each). Both fresh and cryopreserved MSCs showed equivalent number of population doublings. Similarly, doubling time was also comparable in both fresh and cryopreserved MSCs. Colony-forming unit assay on day 14 indicated equivalent percentage plating efficiency [(No. of colonies counted/No. of cells plated) 100] for MSCs harvested from fresh or cryopreserved adipose tissue. (C) Adipogenic induction in fresh and cryopreserved adipose tissueederived MSC cultures (n ¼ 5) was assessed by treating MSCs (fresh and cryopreserved) with adipogenic induction medium for 21days. Differentiation into adipocytes was compared by means of histochemistry, immunofluorescence and RT-PCR. Equivalent number of oil red Oepositive cells and oil red O uptake along with immunofluorescence staining for FABP4 fatty acid binding protein 4 (FABP4) and mRNA levels of lipoprotein lipase (LPL) and peroxisome proliferator-activated-receptor-gamma (PPAR-g) between fresh and cryopreserved adipose tissue-MSCs revealed no significant difference. (D) MSCs from both sources (n ¼ 5) show similar osteogenic induction as detected by von Kossa stain of matrix mineralization deposition (analyzed with the use of Image J software), immunofluorescence staining for osteonectin and quantitative analysis of osteogenic-associated gene expression (osteocalcin and alkaline phosphatase) with RT-PCR. (E) Chondrogenic potential (n ¼ 5) of fresh and cryopreserved MSC cultures was also similar, as indicated by quantification of alcian blue staining and quantitative RT-PCR for mRNA expression of aggrecan and collagen type 2. (F) Number of neuron-like cells, nestin expression as determined by immunofluorescence staining and RT-PCR for the expression of neurogenic specific genes (NSE [neuron-specific-enolase], NFM [neurofilament]) indicated equivalent neurogenic potential in fresh and cryopreserved MSC cultures (n ¼ 5).
that cryopreservation of such cells and tissues (being an experimental intervention) can safely and reproducibly maintain the youthful functional capabilities of the tissues and in turn the cells contained within and therefore can be used as an anti-aging strategy. The processes of cryopreservation and thawing as well as concentration and type of cryoprotectant play an important role in maintaining overall biological characteristics of cells. Therefore, different methods of both cryopreservation and thawing (most commonly slow- and rapid-cooling methods) and type and concentration of cryoprotectant (phosphate buffer saline human albumin, dimethyl sulfoxide, ethylene glycol and sucrose) have been used to get
the desired outcomes such as viability, proliferation and differentiation potential. Although previous reports in the literature are varied, the methodology that we used can successfully cryopreserve cells/tissues in a liquid nitrogen container and later thaw the cells/tissues for clinical use [4,5]. The experiments performed in our laboratory in this regard are very promising (Figure 1). We have collected adipose tissue from donors of various ages, which were processed, cryopreserved and stored frozen in liquid nitrogen for extended periods of time. On thawing, the samples were evaluated for cellular content and viability (in particular, mesenchymal stem cell [MSC] content) as well as for potential utility for
Cryopreservation as an anti-aging strategy regenerative medicine and/or tissue engineering applications. Potential clinical utility was evaluated by use of growth characteristics as well as the capacity to differentiate into mesenchymal and non-mesenchymal lineages. The results indicate that cryopreserved cells maintain morphology and high viability. Fluorescence-activated cell sorting analysis confirmed that cells isolated from both fresh and frozen tissues expressed a similar percentage of cells with MSC markers. Long-term in vitro expansion of fresh and cryopreserved cells indicated no difference in either the number or time of population doublings. In addition, it was observed that cryopreservation does not have a significant effects on senescent features (evaluated at passages 1, 5 and 8) of MSCs. MSCs isolated from fresh and frozen tissue were capable of differentiating along adipogenic, chondrogenic, osteogenic and neurogenic pathways, as confirmed by histology and real-time polymerase chain reaction (RT-PCR) analysis of tissue-specific messenger RNAs (mRNAs). No significant functional differences were observed between MSCs from frozen tissue as compared with fresh tissue [4,5]. Similar experiments have also been performed by preserving isolated cells rather than whole tissues. Overall, these findings indicate that storing the cells of healthy and young donors at ultralow temperatures can maintain their youthful properties. The purpose of this study was to show that longterm cryopreservation does not affect the biological properties of whole tissues or cells contained in it. Stem cell banks offer the opportunity to preserve
3
stem cells in viable conditions for prolonged periods of time until needed by the donor. The patients undergoing liposuction or other cosmetic procedure thus have the chance to preserve some tissue/cells when healthy and young that could be used later for tissue engineering and regenerative medicine applications. Because donor age negatively regulates stem cells, storing the cells at low temperatures can maintain their youthful properties, and therefore the strategy can be used as an anti-aging strategy.
Disclosure of interests: Dr Choudhery does not have financial or non-financial competing interests. Dr Harris is the CSO for Adicyte, Inc. References [1] Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, et al. Stem-cell ageing modified by the cyclin dependent kinase inhibitor p16INK4a. Nature 2006;443: 421e6. [2] Stolzing A, Sethe S, Scutt AM. Stressed stem cells: temperature response in aged mesenchymal stem cells. Stem Cells Dev 2006;15:478e87. [3] Rattan SI, Singh R. Progress and prospects: gene therapy in aging. Gene Ther 2009;16:3e9. [4] Choudhery MS, Badowski M, Muise A, Harris DT. Utility of cryopreserved umbilical cord tissue for regenerative medicine. Curr Stem Cell Res Ther 2013;8:370e80. [5] Choudhery MS, Badowski M, Muise A, Pierce J, David DT. Cryopreservation of whole adipose tissue for future use in regenerative medicine. J Surgical Res 2014;187:24e35.
