DATE CREATED: January 04, 2009
ACTION PLAN
PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)
PAPER TITLE : The Liver Cell Line Derived Conditioned Medium Enhanced Proliferation and Myotube formation of Primary Isolated Rat Cardiomyocytes
A) BACKGROUND:
Cardiomyocytes are pivotal elements of myocardial tissue structure and function. In the field of bioengineering and tissue engineering, many approaches to mimic natural cardiac tissues have been processed. For this purpose, the development of efficient culture condition to enhance cardiomyocyte proliferative activity and myotube formation has been a topic to establish suitable environment to mimic natural cardiac tissues.
B) HYPOTHESIS
In this study, isolated neonatal rat cardiomyocytes were cultured under several culture conditions in vitro, and cell proliferation, myotube formation and functionality were characterized by cell morphology, immunocytochemical staining and time lapse confocal scanning microscopic technique. When cardiomyocytes were cultured in liver cell line (HepG2) derived conditioned medium without any external addition of growth factors and cytokines, highly straitened cell morphology was detected and subsequent fusion into mytotubes was highly developed.
C) SPECIFIC AIMS
AIM 1 - Mimic natural cardiac tissues
AIM 2 - To define effect of HepG2 conditioned medium
D) GENERAL EXPERIMENTAL APPROACH
Primary culture of cardiomyocytes
Cardiac cells were prepared by primary culture of neonate rats. The neonatal SD rats of day 1 were anastesized and carefully open the chest and harvested hearts. The hearts were immersed in heparin containing PBS and trimmed small vessels and connective tissues other than heart muscle. Heart tissues were cut to 4~5 pieces and washed with heparin-PBS to remove red blood cells. Tissue pieces were chopped and digested with collagenase type II for 20 minutes, and isolated cells were separated with cell strainer. This cell isolation process were repeated 4~6 times until all the tissue pieces were digested. Collected cells were preplated in the petri-dish for 30 minutes to seperate the Fibroblast cellls, and suspended cells were recollected by centrifuge.
All the animal protocol were reviewed and approved from Animal Experiment Review Board[proof] and approved as protocol number 04374 of Harvard university.
Collection of HepG2 Conditioned Medium
HepG2 cells (ATCC HB-8605) were cultured in tissue-culture flasks at 37°C in a 5% CO2 humidified incubatorusing DMEM (Gibco) supplemented with 10% FBS, and 1% streptomycin/penicillin (Gibco) at a seeding density of 5.0 x 104 cells/cm2. The culture medium was collected after 4 days of culture, filter-sterilized through a 0.22-um filter, supplemented with 0.1 mM Beta-mercaptoethanol (Sigma), and stored at 4°C prior to use.
E) DESIGN PITFALLS AND ALTERNATIVES
F) ANTICIPATED FIGURES FOR PAPER or when you have data, FIGURES FOR PAPER
Figure 1. The population of isolated primary fetal rat cardiomyocytes, and the morphological and immunocytochemical characterization of rat cardiomyocytes in different culture conditions: (I) FACS analysis for α-Myosin Heavy Chain positive cardiomyocyte population. (II) Morphological observation of cardiomyocyte culture in various culture conditions: G1. Cardiomyocyte culture on preplated cardiac fibroblast layer in normal cardiomyocyte medium. G2. Cardiomyocyte culture on fixated cardiac fibroblast layer in normal cardiomyocyte medium. G3. Cardiomyocyte culture in conditioned medium collected from cardiac fibroblasts culture. G4. Cardiomyocyte culture in 50% conditioned medium collected from HepG2 cell line culture. G5. Cardiomyocyte culture in 100% conditioned medium collected from HepG2 cell line culture. G6. Cardiomyocyte culture in normal cardiomyocyte medium. (III) Immunocytochemical characterization of cardiomyocyte cultures with cardiomyocyte specific antibodies against tropomyosin and sarcomeric α-actinin and with gap junction antibody against connexin 43.
Figure 2. The characterization of the distribution of cardiac fibroblasts and cardiomyocytes, and the proliferation of cardiac fibroblasts in different culture condition and representative morphology of cardiomyocytes: (I) Immunocytochemical staining for the characterization of cardiac fibroblast distribution in various culture condition with fibroblast specific antibody against prolyl 4-hydroxylase and with gap junction antibody against connexin 43. (II) Proliferation assay of cardiac fibroblast in normal cardiomyocyte medium and 100% HepG2 CM. (III) Representative morphology of cariomyocyte in normal cardiomyocyte medium and 100% HepG2 CM.
Figure 3. The confocal scanning microscopic observation of beating mediated calcium fluctuation using calcium indicator (Fluo 4 AM): (I) Time lapsed image of calcium fluctuation in cariomyocyte culture in normal cardiomyocyte medium. (II) Time lapsed image of calcium fluctuation in cariomyocyte culture in 100% HepG2 CM.
Figure 4. Cardiomyocyte proliferation in different culture conditions and the morphological and immunocytochemical characterization of cardiomyocytes with different initial cell seeding densities: (I) Time course image and proliferation assay of cardiomyocytes in normal cardiomyocyte medium and 100% HepG2 CM. (II) Morphological and immunocytochemical evaluation for myotube formation in different cardiomyocyte densities in normal cardiomyocyte medium and 100% HepG2 CM.
Figure 5. The effect of reduced FBS concentration and LIF on cardiomyocyte morphology and cardiomyotube formation: (I) The effect of reduced FBS concentration on myotube formation. (II) The effect of LIF on myotube formation. (III) The effect of IGF II on myotube formation. (IV) The effect of M-CSF on myotube formation. (V) The effect of oncostatin on myotube formation. (VI) The effect of follistatin on myotube formation.
G) FUTURE DIRECTIONS