Optimal in vitro culture conditions for murine predominant immature CD8a+ dendritic cells.
BACKGROUND
The prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells.


METHODS
Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies.


RESULTS
The cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P<0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P<0.05).


CONCLUSIONS
Culture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.
Na
Ning
N
Department of Kidney Transplantation, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China.
Xu
Lin
L
Cao
Kai-yuan
KY
Luo
Yun
Y
Yuan
Guang-qing
GQ
Xiang
Peng
P
Hong
Liang-qing
LQ
Li
Shu-nong
SN
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D016827:CD8 Antigens / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008858:Microscopy, Phase-Contrast
D016827:CD8 Antigens; C409469:CD8 antigen, alpha chain; D016178:Granulocyte-Macrophage Colony-Stimulating Factor