Displaying publications 1 - 20 of 25 in total

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  1. Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study
    Hani H, Ibrahim TA, Othman AM, Lila MA, bt Allaudin ZN
    Xenotransplantation, 2010 12 17;17(6):469-80.
    PMID: 21158948 DOI: 10.1111/j.1399-3089.2010.00616.x
    BACKGROUND: Insufficient availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of caprine as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for caprine islet isolation and purification for culture establishment, and to subsequently assess their viable and functional potential.

    METHODS: Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA.

    RESULTS: The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets.

    CONCLUSION: The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches.

    Matched MeSH terms: Cell Separation/methods*
  2. Purification of Plasmodium and Babesia- infected erythrocytes using a non-woven fabric filter
    Tao ZY, Liu WP, Dong J, Feng XX, Yao DW, Lv QL, et al.
    Trop Biomed, 2020 Dec 01;37(4):911-918.
    PMID: 33612745 DOI: 10.47665/tb.37.4.911
    The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.
    Matched MeSH terms: Cell Separation/methods*
  3. Fulltext Comparative analysis of cardiovascular development related genes in stem cells isolated from deciduous pulp and adipose tissue
    Loo ZX, Kunasekaran W, Govindasamy V, Musa S, Abu Kasim NH
    ScientificWorldJournal, 2014;2014:186508.
    PMID: 25548778 DOI: 10.1155/2014/186508
    Human exfoliated deciduous teeth (SHED) and adipose stem cells (ASC) were suggested as alternative cell choice for cardiac regeneration. However, the true functionability of these cells toward cardiac regeneration is yet to be discovered. Hence, this study was carried out to investigate the innate biological properties of these cell sources toward cardiac regeneration. Both cells exhibited indistinguishable MSCs characteristics. Human stem cell transcription factor arrays were used to screen expression levels in SHED and ASC. Upregulated expression of transcription factor (TF) genes was detected in both sources. An almost equal percentage of >2-fold changes were observed. These TF genes fall under several cardiovascular categories with higher expressions which were observed in growth and development of blood vessel, angiogenesis, and vasculogenesis categories. Further induction into cardiomyocyte revealed ASC to express more significantly cardiomyocyte specific markers compared to SHED during the differentiation course evidenced by morphology and gene expression profile. Despite this, spontaneous cellular beating was not detected in both cell lines. Taken together, our data suggest that despite being defined as MSCs, both ASC and SHED behave differently when they were cultured in a same cardiomyocytes culture condition. Hence, vigorous characterization is needed before introducing any cell for treating targeted diseases.
    Matched MeSH terms: Cell Separation/methods*
  4. Fulltext Optimisation of laboratory procedures for isolating human peripheral blood derived neutrophils
    Maqbool M, Vidyadaran S, George E, Ramasamy R
    Med J Malaysia, 2011 Oct;66(4):296-9.
    PMID: 22299545 MyJurnal
    Functional analysis of neutrophils requires isolation of these cells in the laboratory. Current isolation procedures are time consuming and can potentially activate the resting neutrophils. Thus, in this present study, we have optimised an existing laboratory protocol for human neutrophil isolation from peripheral blood. Twenty ml of blood samples were subjected to optimised density gradient separation and dextran sedimentation to obtain a pure population of neutrophils. The efficacy of the optimised manual post isolation of neutrophils was compared with pre isolation count performed by an automated haematology analyzer. The recovery of neutrophils via our optimised methods was 65.5% in comparison with neutrophils counts at pre-isolation. The morphological analysis of isolated neutrophils indicated the purity level more than 95% using Leishman staining. Our optimised laboratory procedures for neutrophils isolation successfully harvested neutrophils with good viability, purity and post recovery yield. This procedure provides an ideal platform to separate neutrophils for in vitro studies.
    Matched MeSH terms: Cell Separation/methods*
  5. Isolation techniques of murine bone marrow progenitor cells and their adipogenic, neurogenic and osteogenic differentiation capacity
    Ng AM, Kojima K, Kodoma S, Ruszymah BH, Aminuddin BS, Vacanti AC
    Med J Malaysia, 2008 Jul;63 Suppl A:121-2.
    PMID: 19025015
    Bone marrow derived progenitor cells have been widely studied for its multipotent property and have proofed to be an important resource in regenerative medicine. However, the propagation of murine bone marrow appeared to be a great challenge as compared to other mammalian species. In this study, various isolation techniques and the plasticity of the isolated cells were evaluated. Our result shows that magnetic sorting technique yielded the most viable cells and displayed wider differentiation capacity.
    Matched MeSH terms: Cell Separation/methods*
  6. Flow cytometric analysis of intracellular myeloperoxidase distinguishes lymphocytes, monocytes and granulocytes
    Tay SP, Cheong SK, Hamidah NH, Ainoon O
    Malays J Pathol, 1998 Dec;20(2):91-4.
    PMID: 10879268
    A study was undertaken to evaluate the ability of flow cytometric analysis of intracellular myeloperoxidase (MPO) in differentiating populations of lymphocytes (L), monocytes (M) and granulocytes (G), by means of lysed whole blood method. Anticoagulated blood from 23 normal individuals was lysed with FACS lysing solution and permeabilized with FACS permeabilizing solution before subjected to direct immunofluorescence staining. The geometric means of the fluorescence intensity were measured using FACSCalibur flow cytometer (Becton Dickinson). Populations of L, M and G were gated based on their light scatter characteristics and expression of CD14 and CD45. Then, the fluorescence intensity of MPO expression was studied in these individual cell populations. The results showed that fluorescence intensity of MPO was the strongest in G and weakest in L, whereas M showed intermediate fluorescence intensity. Our findings reveal that discrimination of these three cell types is achievable based upon the sole expression of intracellular MPO.
    Matched MeSH terms: Cell Separation/methods*
  7. A comparative study of two methods for the isolation of human leucocytes for DNA extraction
    Lim LH, Ton SH, Cheong SK
    Malays J Pathol, 1990 Jun;12(1):39-41.
    PMID: 2090888
    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.
    Matched MeSH terms: Cell Separation/methods*
  8. Fulltext Cell patterning for liver tissue engineering via dielectrophoretic mechanisms
    Yahya WN, Kadri NA, Ibrahim F
    Sensors (Basel), 2014 Jul 02;14(7):11714-34.
    PMID: 24991941 DOI: 10.3390/s140711714
    Liver transplantation is the most common treatment for patients with end-stage liver failure. However, liver transplantation is greatly limited by a shortage of donors. Liver tissue engineering may offer an alternative by providing an implantable engineered liver. Currently, diverse types of engineering approaches for in vitro liver cell culture are available, including scaffold-based methods, microfluidic platforms, and micropatterning techniques. Active cell patterning via dielectrophoretic (DEP) force showed some advantages over other methods, including high speed, ease of handling, high precision and being label-free. This article summarizes liver function and regenerative mechanisms for better understanding in developing engineered liver. We then review recent advances in liver tissue engineering techniques and focus on DEP-based cell patterning, including microelectrode design and patterning configuration.
    Matched MeSH terms: Cell Separation/methods*
  9. Fulltext A hybrid-membrane migration method to isolate high-purity adipose-derived stem cells from fat tissues
    Higuchi A, Wang CT, Ling QD, Lee HH, Kumar SS, Chang Y, et al.
    Sci Rep, 2015;5:10217.
    PMID: 25970301 DOI: 10.1038/srep10217
    Human adipose-derived stem cells (hADSCs) exhibit heterogeneous characteristics, indicating various genotypes and differentiation abilities. The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. We developed a hybrid-membrane migration method that purifies hADSCs from a fat tissue solution with extremely high purity and pluripotency. A primary fat-tissue solution was permeated through the porous membranes with a pore size from 8 to 25 μm, and the membranes were incubated in cell culture medium for 15-18 days. The hADSCs that migrated from the membranes contained an extremely high percentage (e.g., >98%) of cells positive for mesenchymal stem cell markers and showed almost one order of magnitude higher expression of some pluripotency genes (Oct4, Sox2, Klf4 and Nanog) compared with cells isolated using the conventional culture method.
    Matched MeSH terms: Cell Separation/methods*
  10. Fulltext Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods
    Lin HR, Heish CW, Liu CH, Muduli S, Li HF, Higuchi A, et al.
    Sci Rep, 2017 01 10;7:40069.
    PMID: 28071738 DOI: 10.1038/srep40069
    Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not.
    Matched MeSH terms: Cell Separation/methods*
  11. Comparative study on human and bovine AT-SC isolation methods
    Reshak AH, Shahimin MM, Buang F
    Prog Biophys Mol Biol, 2013 Nov;113(2):295-8.
    PMID: 24080186 DOI: 10.1016/j.pbiomolbio.2013.09.001
    Mammalian adipose tissue derived stem cells (AT-SC) have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 h and 2 h under 37 °C in 5% CO2 incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 h under 37 °C in CO2 incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 h under 37 °C in CO2 incubator.
    Matched MeSH terms: Cell Separation/methods*
  12. Isolation and Characterization of Cancer Stem Cells of the Non-Small-Cell Lung Cancer (A549) Cell Line
    Halim NHA, Zakaria N, Satar NA, Yahaya BH
    Methods Mol Biol, 2016;1516:371-388.
    PMID: 27032945 DOI: 10.1007/7651_2016_326
    Cancer is a major health problem worldwide. The failure of current treatments to completely eradicate cancer cells often leads to cancer recurrence and dissemination. Studies have suggested that tumor growth and spread are driven by a minority of cancer cells that exhibit characteristics similar to those of normal stem cells, thus these cells are called cancer stem cells (CSCs). CSCs are believed to play an important role in initiating and promoting cancer. CSCs are resistant to currently available cancer therapies, and understanding the mechanisms that control the growth of CSCs might have great implications for cancer therapy. Cancer cells are consist of heterogeneous population of cells, thus methods of identification, isolation, and characterisation of CSCs are fundamental to obtain a pure CSC populations. Therefore, this chapter describes in detail a method for isolating and characterizing a pure population of CSCs from heterogeneous population of cancer cells and CSCs based on specific cell surface markers.
    Matched MeSH terms: Cell Separation/methods*
  13. 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
  14. 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*
  15. 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*
  16. 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*
  17. 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*
  18. 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
  19. 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
  20. 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
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