Lymphocyte cultures with small numbers of cells

Prior experience with cultures of cerebrospinal fluid lymphocytes indicated a need to develop methods for culturing small numbers of cells. Peripheral blood lymphocytes (PBL) were obtained from 20 normal volunteers. Standard microcultures using 100 X 10(3) PBL/0.2 ml and cultures with 50, 25 and 12.5 X 10(3) in 0.1 ml or 0.2 ml were established in RPMI 1640 with autologous plasma. These cultures were incubated with PHA (1--30 microgram) for 3, 4 and 5 days, pulsed with [3H]thymidine and harvested. In unstimulated cultures, cpm declined linearly with decreasing cell numbers. Standard cultures (100 X 10(3) PBL/0.2 ml) had maximal PHA stimulation (80,916 +/- 6394) at day 3 with 30 microgram PHA. Other 0.2 ml cultures had lower cpm. By culturing 25 X 10(3) PBL in 0.1 ml for 3 days cpm were 82,874 +/- 6875 with 30 microgram PHA and 77,153 +/- 6022 with 15 microgram PHA and were similar to standard cultures. Similar cpm were seen with 12.5 X 10(3) PBL in 0.1 ml after 4 days with 30 micrograms of PHA (80,838 +/- 6674) and with 15 micrograms of PHA (72,860 +/- 6243), and also after 5 days with 30 micrograms of PHA (86,703 +/- 6732) and with 15 micrograms of PHA (74,066 +/- 6388). The maximal response (126,578 +/- 6580) was seen with 25 X 10(3) PBL/0.1 ml at day 4 with 30 micrograms of PHA. By decreasing culture volume to 0.1 ml and increasing time, the number of cells necessary to give PHA responses similar to standard cultures can be reduced by 75--88%.