Comparison of fetal bovine serum and platelet-rich plasma on human lipoaspirate-derived mesenchymal stem cell proliferation.

Background: Lipoaspirate-derived stem cells (LSCs) are very promising for regenerative medicine, e.g. to treat acute myocard infarction. Fetal bovine serum (FBS) is commonly used to propagate the LSCs. However, for its clinical application, FBS contains xeno-proteins that are potential to elicit immune rejection in patients. Platelet rich plasma (PRP) is one of the candidates to replace FBS. This study was aimed to compare the proliferation of LSCs cultured in 5% PRP, 10% PRP, and FBS containing medium (MesenCult®). Methods: LSCs were cultured in 5% PRP/DMEM, 10% PRP/DMEM, and MesenCult®. After the primary culture reached its confluency, cells were harvested using TrypLE Select and seeded (around 20,000 viable cells) in new vessels in the same media. Passages were done until passage-5, with six replications. Population doubling time (PDT) of the three groups were analyzed using Kruskal Wallis test. Results: LSCs showed different proliferation rates when cultured in 5% PRP/DMEM, 10% PRP/DMEM, and MesenCult®. PDT of the three experimental groups in passage 1-5 were significanly different (p < 0.05), with the lowest rank was cultured in medium of 10% PRP/DMEM. Conclusion: The results suggest that 10% PRP/DMEM can be used as an alternative to replace FBS in LSC culture.

Effect of adipose tissue processing procedures in culture result: a study preliminary.

Background: There are various methods of processing adipose tissue before culture, depending on the adipose tissue samples. The aim of this study is to compare several modifications of culturing and sub-culturing procedures of adipose tissue to fit the condition in our laboratory. Method: This is a descriptive study that was done in the Immunology and Endocrinology Integrated Laboratory, University of Indonesia, from October 2009 to April 2010. Three adipose tissue processing procedures, various amount of seeding and two subculture methods were compared in term of cell yield and time needed. In the first procedure, collagenase-1 digestion was done in 30minutes, cell seeding were 24,000 and 36,000 per flask; in the second procedure, collagenase-1 digestion was done in 60minutes, cell seeding were 24,000, 48,000, and 72,000 per flask; and in the third procedure, the adipose tissue remnants from the first procedure were again digested for another 45 minutes, cell seeding were 74,000, and 148,000 per flask. Difference in subculture methods were the presence or absence of washing step. Result: Procedure 1 yielded the lowest amount of cell, and after culture, the cells grew very slow, and was contaminated before harvest of primary culture. Procedure-2 and -3 succeeded to yield primary cultures. Some of the cultures were contaminated, so that further subculture was not applicable, and only one tissue processing procedure (procedure 2: 60 minute collagenase-1 digestion, without lysis buffer, cell seeding 48,000 and 72,000) could complete the three subcultures. Though some of the procedures could not be completed, final result could be concluded. Conclusion: In this preliminary study, 60 minute colagenase-1 digestion with intermittent shaking every 5 minutes and cell seeding around 50,000 or more, followed by subculture method without washing step gave the best result.

Cytology technique: development of a simple spot method for cultured cell suspension.

Aim To develop a simple spot method to attach cultured cells in suspension on to a glass slide. Methods We compared three approaches using both conventional and special glass slide (Shandon-Polysin)., either without additional fetal bovine serum (FBS), or with addition of 3 or 10 μl of FBS to a 20 μl sample (altogether there were six approaches). The slides were examined qualitatively for the background color, boundary color and intactness, and whether there were folded and detached parts. Further, for each slide, the attached intact cells were counted, and the percentage of attached intact cells per number of spotted cells was calculated. The difference in attach intact cells between different approaches was analyzed by ANOVA using SPSS 13.0 for windows. Results There were no significant difference in the percentage of attached intact cells between the six approaches (P= 0.804), though the approach using special glass slide without additional FBS (FBS final concentration 5%) yield the highest percentage of attached intact cells, showed clean background without folded parts. Conclusions We have developed a simple spot method for cultured cell suspension, and the best approach to make spot specimen is using special glass slide with 5% FBS in the cell suspension.

Comparison of various procedures in the processing of binucleated-lymphocytes on to glass slide to detect chromosomal breakage.

Binucleated lymphocytes can be screened for micronuclei to assess chromosomal damage. There are various procedures to get slides containing binucleated lymphocytes, that are different in harvesting, fixation, and slide preparation methods. Screening binucleated lymphocytes to find a micronucleus needs at least 800 cells with intact cytoplasm. This study aimed to analyze the various procedures and simplified procedures to know which procedure gave the most abundant binucleated lymphocytes with intact cytoplasm and best staining properties for the purpose of micronucleus scoring. Seven heparinized blood samples were obtained from the Dept. of Obstetrics and gynecology, Faculty of medicine, University of Indonesia, Jakarta. The 7 blood samples were subjected to 17 procedures different in harvesting (with or without washing), slide preparation (smear and spot method, and using a cytocentrifuge), and fixation methods (methanol for 1 minute, methanol brief, methanol/glacial acetic acid 3:1 or 9:1). Our results showed that fixatives containing glacial acetic acid are not suitable for micronucleus test. To generate binucleated lymphocytes with intact cytoplasm as much as possible, the procedure should be conducted without washing steps. Methanol fixation either briefly or 1 minute is preferable, and for the ease of screening cytocentrifuge preparation, followed by spot method is preferable.