Displaying publications 21 - 40 of 47 in total

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  1. Isolation, Expansion, and Characterization of Wharton's Jelly-Derived Mesenchymal Stromal Cell: Method to Identify Functional Passages for Experiments
    Aung SW, Abu Kasim NH, Ramasamy TS
    Methods Mol Biol, 2019;2045:323-335.
    PMID: 31201682 DOI: 10.1007/7651_2019_242
    The therapeutic potential of human mesenchymal stromal stem cells (hMSCs) for cell-based therapeutic is greatly influenced by the in vitro culture condition including the culture conditions. Nevertheless, there are many technical challenges needed to be overcome prior to the clinical use including the quantity, quality, and heterogeneity of the cells. Therefore, it is necessary to develop a stem cell culture procedure or protocol for cell expansion in order to generate reproducible and high-quality cells in accordance with good manufacturing practice for clinical and therapeutic purposes. Here we assessed the MSCs characteristic of human Wharton's jelly mesenchymal stromal cells in in vitro culture according to the criteria established by the International Society for Cellular Therapy. Besides, the viability of the WJMSCs was determined in order to increase the confidence that the cells are employed to meet the therapeutic efficacy.
    Matched MeSH terms: Cell Separation/methods
  2. Development and characteristics of polymer monoliths for advanced LC bioscreening applications: A review
    Acquah C, Moy CKS, Danquah MK, Ongkudon CM
    J Chromatogr B Analyt Technol Biomed Life Sci, 2016 Mar 15;1015-1016:121-134.
    PMID: 26919447 DOI: 10.1016/j.jchromb.2016.02.016
    Biomedical research advances over the past two decades in bioseparation science and engineering have led to the development of new adsorbent systems called monoliths, mostly as stationary supports for liquid chromatography (LC) applications. They are acknowledged to offer better mass transfer hydrodynamics than their particulate counterparts. Also, their architectural and morphological traits can be tailored in situ to meet the hydrodynamic size of molecules which include proteins, pDNA, cells and viral targets. This has enabled their development for a plethora of enhanced bioscreening applications including biosensing, biomolecular purification, concentration and separation, achieved through the introduction of specific functional moieties or ligands (such as triethylamine, N,N-dimethyl-N-dodecylamine, antibodies, enzymes and aptamers) into the molecular architecture of monoliths. Notwithstanding, the application of monoliths presents major material and bioprocess challenges. The relationship between in-process polymerisation characteristics and the physicochemical properties of monolith is critical to optimise chromatographic performance. There is also a need to develop theoretical models for non-invasive analyses and predictions. This review article therefore discusses in-process analytical conditions, functionalisation chemistries and ligands relevant to establish the characteristics of monoliths in order to facilitate a wide range of enhanced bioscreening applications. It gives emphasis to the development of functional polymethacrylate monoliths for microfluidic and preparative scale bio-applications.
    Matched MeSH terms: Cell Separation
  3. The use of sonication treatment to decellularize aortic tissues for preparation of bioscaffolds
    Azhim A, Syazwani N, Morimoto Y, Furukawa KS, Ushida T
    J Biomater Appl, 2014 Jul;29(1):130-41.
    PMID: 24384523 DOI: 10.1177/0885328213517579
    A novel decellularization method using sonication treatment is described. Sonication treatment is the combination of physical and chemical agents. These methods will disrupt cell membrane and release cell contents to external environments. The cell removal was facilitated by subsequent rinsing of sodium dodecyl sulfate detergents. Sonication treatment is used in the preparation of complete decellularized bioscaffolds. The aim of this study is to confirm the usefulness of sonication treatment for preparation of biological scaffolds. In this study, samples of aortic tissues are decellularized by sonication treatment at frequency of 170 kHz in 0.1% and 2% sodium dodecyl sulfate detergents for 10-h treatment time. The relation between decellularization and sonication parameters such as dissolved oxygen concentration, conductivity, and pH is investigated. Histological analysis and biomechanical testing is performed to evaluate cell removal efficiency as well as changes in biomechanical properties. Minimal inflammation response elicit by bioscaffolds is confirmed by xenogeneic implantation and immunohistochemistry. Sonication treatment is able to produce complete decellularized tissue suggesting that these treatments could be applied widely as one of the decellularization method.
    Matched MeSH terms: Cell Separation
  4. Fulltext Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population
    Farea M, Halim AS, Abdullah NA, Lim CK, Mokhtar KI, Berahim Z, et al.
    Int J Mol Sci, 2013;14(6):11157-70.
    PMID: 23712356 DOI: 10.3390/ijms140611157
    Hertwig's epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.
    Matched MeSH terms: Cell Separation/methods*
  5. Morphological changes of post-isolation of caprine pancreatic islet
    Hani H, Allaudin ZN, Tengku Ibrahim TA, Mohd-Lila MA, Sarsaifi K, Camalxaman SN, et al.
    In Vitro Cell Dev Biol Anim, 2015 Feb;51(2):113-20.
    PMID: 25303943 DOI: 10.1007/s11626-014-9821-7
    Pancreatic islet transplantation is commonly used to treat diabetes. Cell isolation and purification methods can affect the structure and function of the isolated islet cells. Thus, the development of cell isolation techniques that preserve the structure and function of pancreatic islet cells is essential for enabling successful transplantation procedures. The impact of purification procedures on cell function can be assessed by performing ultrastructure and in vivo studies. Thus, the aim of this study was to evaluate the effect of caprine islets purification procedure on islet cell ultrastructure and functional integrity prior to and post-isolation/purification. The islets were isolated from caprine pancreas by using an optimized collagenase XI-S concentration, and the cells were subsequently purified using Euro-Ficoll density gradient. In vitro viability of islets was determined by fluorescein diacetate and propidium iodide staining. Static incubation was used to assess functionality and insulin production by islet cells in culture media when exposed to various levels of glucose. Pancreatic tissues were examined by using light microscopy, fluorescence microscopy, scanning, and transmission electron microscopy. In vivo viability and functionality of caprine islets were assessed by evaluating the transplanted islets in diabetic mice. Insulin assay of glucose-stimulated insulin secretion test showed that the insulin levels increased with increasing concentration of glucose. Thus, purified islets stimulated with high glucose concentration (25 mM) secreted higher levels of insulin (0.542 ± 0.346 μg/L) than the insulin levels (0.361 ± 0.219, 0.303 ± 0.234 μg/L) secreted by exposure to low glucose concentrations (1.67 mM). Furthermore, insulin levels of recipient mice were significantly higher (p cell integrity of peripheral region, the alterations did not significantly alter the functionality and viability of the purified islets.
    Matched MeSH terms: Cell Separation/methods*
  6. Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly
    Salehinejad P, Alitheen NB, Ali AM, Omar AR, Mohit M, Janzamin E, et al.
    In Vitro Cell Dev Biol Anim, 2012 Feb;48(2):75-83.
    PMID: 22274909 DOI: 10.1007/s11626-011-9480-x
    Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.
    Matched MeSH terms: Cell Separation/methods*
  7. Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells
    Wee AS, Lim CK, Merican AM, Ahmad TS, Kamarul T
    In Vitro Cell Dev Biol Anim, 2013 Jun;49(6):424-32.
    PMID: 23708918 DOI: 10.1007/s11626-013-9626-0
    In vitro cellular proliferation and the ability to undergo multilineage differentiation make bone marrow-derived multipotent stromal cells (MSCs) potentially useful for clinical applications. Several methods have been described to isolate a homogenous bone marrow-derived MSCs population; however, none has been proven most effective, mainly due to their effects on proliferation and differentiation capability of the isolated cells. It is hypothesized that our newly established total cell pooling method may provide a better alternative as compared to the standard isolation method (density gradient centrifugation method). For the total cell pooling method, MSCs were isolated from rabbit bone marrow and were subsequently cultured in the growth medium without further separation as in the standard isolation method. The total cell pooling method was 65 min faster than the standard isolation method in completing cell isolation. Nevertheless, both methods did not differ significantly in the number of primary viable cells and population doubling time in the cultures (p > 0.05). The isolated cells from both methods expressed CD29 and CD44 markers, but not CD45 markers. Furthermore, they displayed multilineage differentiation characteristics of chondroblasts, osteoblasts, and adipocytes. In conclusion, both methods provide similar efficiency in the isolation of rabbit bone marrow-derived MSCs; however, the total cell pooling method is technically simpler and more cost effective than the standard isolation method.
    Matched MeSH terms: Cell Separation/methods*
  8. Value of human amniotic epithelial cells in tissue engineering for cornea
    Fatimah SS, Ng SL, Chua KH, Hayati AR, Tan AE, Tan GC
    Hum. Cell, 2010 Nov;23(4):141-51.
    PMID: 21166885 DOI: 10.1111/j.1749-0774.2010.00096.x
    Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.
    Matched MeSH terms: Cell Separation
  9. Long-term evaluation of osteoarthritis sheep knee, treated with TGF-β3 and BMP-6 induced multipotent stem cells
    Ude CC, Shamsul BS, Ng MH, Chen HC, Ohnmar H, Amaramalar SN, et al.
    Exp Gerontol, 2018 04;104:43-51.
    PMID: 29421350 DOI: 10.1016/j.exger.2018.01.020
    BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery.

    METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography.

    RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance.

    CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-β3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.

    Matched MeSH terms: Cell Separation
  10. A correlation of conductivity medium and bioparticle viability on dielectrophoresis-based biomedical applications
    Deivasigamani R, Mohd Maidin NN, Abdul Nasir NS, Abdulhameed A, Ahmad Kayani AB, Mohamed MA, et al.
    Electrophoresis, 2023 Mar;44(5-6):573-620.
    PMID: 36604943 DOI: 10.1002/elps.202200203
    Dielectrophoresis (DEP) bioparticle research has progressed from micro to nano levels. It has proven to be a promising and powerful cell manipulation method with an accurate, quick, inexpensive, and label-free technique for therapeutic purposes. DEP, an electrokinetic phenomenon, induces particle movement as a result of polarization effects in a nonuniform electrical field. This review focuses on current research in the biomedical field that demonstrates a practical approach to DEP in terms of cell separation, trapping, discrimination, and enrichment under the influence of the conductive medium in correlation with bioparticle viability. The current review aims to provide readers with an in-depth knowledge of the fundamental theory and principles of the DEP technique, which is influenced by conductive medium and to identify and demonstrate the biomedical application areas. The high conductivity of physiological fluids presents obstacles and opportunities, followed by bioparticle viability in an electric field elaborated in detail. Finally, the drawbacks of DEP-based systems and the outlook for the future are addressed. This article will aid in advancing technology by bridging the gap between bioscience and engineering. We hope the insights presented in this review will improve cell suspension medium and promote DEP-viable bioparticle manipulation for health-care diagnostics and therapeutics.
    Matched MeSH terms: Cell Separation
  11. New method for the isolation of endothelial cells from large vessels
    Ataollahi F, Pingguan-Murphy B, Moradi A, Wan Abas WA, Chua KH, Abu Osman NA
    Cytotherapy, 2014 Aug;16(8):1145-52.
    PMID: 24831838 DOI: 10.1016/j.jcyt.2014.01.010
    Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.
    Matched MeSH terms: Cell Separation
  12. In vitro expression of erythropoietin by transfected human mesenchymal stromal cells
    Mok PL, Cheong SK, Leong CF, Othman A
    Cytotherapy, 2008;10(2):116-24.
    PMID: 18368590 DOI: 10.1080/14653240701816996
    Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro.
    Matched MeSH terms: Cell Separation
  13. Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells
    Liau LL, Ruszymah BHI, Ng MH, Law JX
    Curr Res Transl Med, 2020 01;68(1):5-16.
    PMID: 31543433 DOI: 10.1016/j.retram.2019.09.001
    Mesenchymal stromal cells (MSCs) are widely used in the clinic because they involve fewer ethical issues and safety concerns compared to other stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). MSCs derived from umbilical cord Wharton's jelly (WJ-MSCs) have excellent proliferative potential and a faster growth rate and can retain their multipotency for more passages in vitro compared to adult MSCs from bone marrow or adipose tissue. WJ-MSCs are used clinically for repairing tissue injuries of the spinal cord, liver and heart with the aim of regenerating tissue. On the other hand, WJ-MSCs are also used clinically to ameliorate immune-mediated diseases based on their ability to modulate immune responses. In the field of tissue engineering, WJ-MSCs capable of differentiating into multiple cell lineages have been used to produce a variety of engineered tissues in vitro that can then be transplanted in vivo. This review discusses the characteristics of WJ-MSCs, the differences between WJ-MSCs and adult MSCs, clinical studies involving WJ-MSCs and future perspectives of WJ-MSC research and clinical applications. To summarize, WJ-MSCs have shown promise in treating a variety of diseases clinically. However, most clinical trials/studies reported thus far are relatively smaller in scale. The collected evidence is insufficient to support the routine use of WJ-MSC therapy in the clinic. Thus, rigorous clinical trials are needed in the future to obtain more information on WJ-MSC therapy safety and efficacy.
    Matched MeSH terms: Cell Separation/methods
  14. Generation of mesenchymal stem cell from human umbilical cord tissue using a combination enzymatic and mechanical disassociation method
    Tong CK, Vellasamy S, Tan BC, Abdullah M, Vidyadaran S, Seow HF, et al.
    Cell Biol Int, 2011 Mar;35(3):221-6.
    PMID: 20946106 DOI: 10.1042/CBI20100326
    MSCs (mesenchymal stem cells) promise a great potential for regenerative medicine due to their unique properties of self-renewal, high plasticity, modulation of immune response and the flexibility for genetic modification. Therefore, the increasing demand for cellular therapy necessitates a larger-scale production of MSC; however, the technical and ethical issues had put a halt on it. To date, studies have shown that MSC could be derived from human UC (umbilical cord), which is once considered as clinical waste. We have compared the two conventional methods which are classic enzymatic digestion and explant method with our newly tailored enzymatic-mechanical disassociation method to generate UC-MSC. The generated UC-MSCs from the methods above were characterized based on their immunophenotyping, early embryonic transcription factors expression and mesodermal differentiation ability. Our results show that enzymatic-mechanical disassociation method increase the initial nucleated cell yield greatly (approximately 160-fold) and maximized the successful rate of UC-MSC generation. Enzymatic-mechanical disassociation-derived UC-MSC exhibited fibroblastic morphology and surface markers expression of CD105, CD73, CD29, CD90 and MHC class I. Furthermore, these cells constitutively express early embryonic transcription factors (Nanog, Oct-4, Sox-2 and Rex-1), as confirmed by RT-PCR, indicating their multipotency and high self-renewal capacity. They are also capable of differentiating into osteoblasts and adipocytes when given an appropriate induction. The present study demonstrates a new and efficient approach in generating MSC from UC, hence serving as ideal alternative source of mesenchymal stem cell for clinical and research use.
    Matched MeSH terms: Cell Separation/methods
  15. A plethora of human pluripotent stem cells
    Gao L, Thilakavathy K, Nordin N
    Cell Biol Int, 2013 Sep;37(9):875-87.
    PMID: 23619972 DOI: 10.1002/cbin.10120
    At the early stages of mammalian development, a number of developmentally plastic cells appear that possess the ability to give rise to all of the differentiated cell types normally derived from the three primary germ layers - unique character known as pluripotency. To date, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been shown to be truly pluripotent. However, recent studies have revealed a variety of other cells that demonstrate pluripotentiality, including very small embryonic-like stem cells (VSELs), amniotic fluid stem cells (AFSCs), marrow-isolated adult multilineage inducible cells (MIAMI) and multipotent adult precursor cells (MAPCs). This review summarises key features of these six kinds of pluripotent and potentially pluripotent stem cells (ESCs, iPSCs, VSELs, AFSCs, MIAMI and MAPCs) and the evidence for their pluripotency properties.
    Matched MeSH terms: Cell Separation
  16. Isolation and characterization of primary microglia from post-natal murine brain tissues: a comparison of two methods
    Jose S, Tan SW, Tong CK, Vidyadaran S
    Cell Biol Int, 2015 Dec;39(12):1355-63.
    PMID: 26194799 DOI: 10.1002/cbin.10516
    Microglia are resident macrophages of the central nervous system (CNS). Apart from playing vital roles as sentinel cells, they are crucial in physiological processes such as synaptic pruning during brain development. CNS disorders require an understanding of the contribution of each cellular compartment to the pathogenesis. Elucidating the role of microglia in disease development and progression in the intricate CNS environment is technically challenging and requires the establishment of reliable, reproducible techniques to isolate and culture microglia. A number of different protocols have been developed for isolation of neonatal microglia and here we compare two widely used methods, namely, mild trypsinization and EasySep® magnetic separation. EasySep® magnetic separation provided higher microglia yield, and flow cytometric evaluation of CD11b and F4/80 markers revealed that EasySep® separation method also produced significantly higher purity compared to mild trypsinization. Microglia isolated using EasySep® separation method were functional, as demonstrated by the generation of nitric oxide, IL-6, TNF-α, and MCP-1 in response to lipopolysaccharide stimulation. In summary, this study has revealed that magnetic separation is superior to mild trypsinization in terms of yield and purity of microglia.
    Matched MeSH terms: Cell Separation/methods
  17. Guided evaluation and standardisation of mesenchymal stem cell culture conditions to generate conditioned medium favourable to cardiac c-kit cell growth
    Ng WH, Umar Fuaad MZ, Azmi SM, Leong YY, Yong YK, Ng AMH, et al.
    Cell Tissue Res, 2019 Feb;375(2):383-396.
    PMID: 30232595 DOI: 10.1007/s00441-018-2918-7
    Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25 mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.
    Matched MeSH terms: Cell Separation
  18. Alteration of gene expression levels during osteogenic induction of human adipose derived stem cells in long-term culture
    Safwani WK, Makpol S, Sathapan S, Chua KH
    Cell Tissue Bank, 2013 Jun;14(2):289-301.
    PMID: 22476937 DOI: 10.1007/s10561-012-9309-1
    Adipose tissue is a source of multipotent stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic and adipogenic cells. Most studies on human adipose-derived stem cells (ASCs) have been carried out at the early passages. For clinical usage, ASCs need to be expanded in vitro for a period of time to get sufficient cells for transplantation into patients. However, the impact of long-term culture on ASCs molecular characteristics has not been established yet. Several studies have also shown that osteogenic and adipogenic cells have the ability to switch pathways during in vitro culture as they share the same progenitor cells. This data is important to ensure their functionality and efficacy before being used clinically in the treatment of bone diseases. Therefore, we aim to investigate the effect of long-term culture on the adipogenic, stemness and osteogenic genes expression during osteogenic induction of ASCs. In this study, the molecular characteristics of ASCs during osteogenic induction in long-term culture was analysed by observing their morphological changes during induction, analysis of cell mineralization using Alizarin Red staining and gene expression changes using quantitative RT-PCR. Morphologically, cell mineralization at P20 was less compared to P5, P10 and P15. Adipogenesis was not observed as negative lipid droplets formation was recorded during induction. The quantitative PCR data showed that adipogenic genes expression e.g. LPL and AP2 decreased but PPAR-γ was increased after osteogenic induction in long-term culture. Most stemness genes decreased at P5 and P10 but showed no significant changes at P15 and P20. While most osteogenic genes increased after osteogenic induction at all passages. When compared among passages after induction, Runx showed a significant increased at P20 while BSP, OSP and ALP decreased at later passage (P15 and P20). During long-term culture, ASCs were only able to differentiate into immature osteogenic cells.
    Matched MeSH terms: Cell Separation/methods
  19. Human amnion as a novel cell delivery vehicle for chondrogenic mesenchymal stem cells
    Tan SL, Sulaiman S, Pingguan-Murphy B, Selvaratnam L, Tai CC, Kamarul T
    Cell Tissue Bank, 2011 Feb;12(1):59-70.
    PMID: 19953328 DOI: 10.1007/s10561-009-9164-x
    This study investigates the feasibility of processed human amnion (HAM) as a substrate for chondrogenic differentiation of mesenchymal stem cells (MSCs). HAM preparations processed by air drying (AD) and freeze drying (FD) underwent histological examination and MSC seeding in chondrogenic medium for 15 days. Monolayer cultures were used as control for chondrogenic differentiation and HAMs without cell seeding were used as negative control. Qualitative observations were made using scanning electron microscopy analysis and quantitative analyses were based on the sulfated glycosaminoglycans (GAG) assays performed on day 1 and day 15. Histological examination of HAM substrates before seeding revealed a smooth surface in AD substrates, while the FD substrates exhibited a porous surface. Cell attachment to AD and FD substrates on day 15 was qualitatively comparable. GAG were significantly highly expressed in cells seeded on FD HAM substrates. This study indicates that processed HAM is a potentially valuable material as a cell-carrier for MSC differentiation.
    Matched MeSH terms: Cell Separation
  20. Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold
    Mazlyzam AL, Aminuddin BS, Fuzina NH, Norhayati MM, Fauziah O, Isa MR, et al.
    Burns, 2007 May;33(3):355-63.
    PMID: 17321690
    Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.
    Matched MeSH terms: Cell Separation
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