Long Term Culture of Primary AML and CML Cells: Effects of Growth Factor Composition on Cell Expansion and Differentiation
Authors: Venglár Ondrej , Culen Martin , Cerna Jana , Kosarova Zdenka , Mayer Jiri , Racil Zdenek 
Published: October 2019

Abstract
Introduction:
In vitro experiments with primary acute and chronic myeloid leukemia (AML, CML) cells are widely applied for testing of anti-tumor drug activity. Several studies also documented long-term maintenance of primary cells with the support of stromal cells and growth factors. However, to our knowledge, none of these studies sufficiently analyzed the differentiation and phenotype changes occurring during the long-term culture.
Aim:
To evaluate the expansion and phenotype changes of primary AML and CML cells in long-term culture using different cytokine cocktails.
Methods:
Primary leukocytes from 6 de novo AML samples, 5×10^5 per well, (various French-American-British classification) and CD34+ cells isolated from 4 de novo CML samples, 15-50×10^3 per well, were seeded onto a confluent layer of human bone-marrow mesenchymal stromal cells (MSCs) in 24-well plates. Four culture conditions were tested: a) "stem cell" supporting condition, designated as C4, with 4 early acting cytokines SCF, TPO, FLT3-ligand and IL-6, b) myeloid cell supporting condition C6, with the C4 cytokines plus two additional late acting growth factors IL-1 and IL-3, c) myeloid cell supporting condition C7 with the same cytokines as C6 plus terminal differentiation factor GM-CSF, d) condition adopted from previous studies, designated as C3, containing cytokines TPO, IL-3, G-SCF. Additional negative control condition C-neg did not include MSCs nor any cytokines. Experiments were analyzed after 2-weeks of culture. Phenotypization was performed both pre- and post-culture by flow cytometry.
Results:
For all AML samples, the tested culture conditions showed successful maintenance of cells in vitro, although with highly heterogeneous expansion rates for the individual samples (0-14 fold expansion, yielding up to 3.5×10^6 cells per well). No AML cells survived in the negative control condition C-neg, for 4/6 samples. The maximum expansion (representing relative yield of 100%) was obtained with the myelo-condition C7 in 5/6 patients. The mean relative yield from all patients for C7 was 97%. The other conditions provided mean relative yields of: C6 - 79%, C4 - 43%, C3 - 44%, and C-neg - 3.5%.
Comparison of pre- and post-culture phenotypes revealed differentiation of AML cells in vitro, demonstrated by the decrease of primitive CD34+ fraction and shifting of less differentiated CD33dim cells into mature CD33bright fraction (mean log2 reduction in CD34+ cells: C4 - 0.5×, p=0.4, C7 - 1.4×, p=0.01, C6 - 1.3×, p=0.01, C3 - 1.0×, p=0.03; mean log2 reduction in CD33dim cells: C4 - 0.8×, p=0.2, C7 - 2.0×, p<0.001, C6 - 2.0×, p<0.001, C3 - 1.1×, p=0.07). Expression of CD14 and also CD117 showed large intra-patient differences (both increase and decrease), without statistically significant differences between the culture conditions. CD123 expression remained stable.
The CML samples were cultured only with C4, C6, C3 conditions, without the terminal differentiation C7 condition. Expansion reached 33-260 fold; yielding up to 3.3×10^6 cells per well. C3 condition provided the highest yields (mean relative yields: C4 - 29%, C6 - 68%, C3 - 93%, and C-neg - 0%). All three tested conditions produced dominant fraction of more mature myeloid CD33+ cells (mean, 86-94%). Nevertheless, the C4 condition provided the highest proportion of CD34+ cells and produced the least mature CD14+ monocytes, compared to C6 and C3 (mean, CD34+ cell fraction of leukocytes: C4 - 31.7%, C6 - 6.8%, C3 - 4.5%; mean, CD14+ cell fraction of leukocytes: C4 - 6%, C6 - 21%, C3 - 9.6%).
Conclusions:
All tested culture conditions were capable of maintaining both primary AML and CML cells from all samples, during the 2-week culture. The highest in vitro expansion was achieved with conditions using the late-acting cytokines which however induced the highest cell differentiation as well. The C3 condition reported in previous studies also provided insufficient conservation of the original phenotypes. In this setup, the best preservation of original phenotypes and more primitive cells was achieved using the C4 "stem cell" condition containing early acting cytokines.
Supported by Ministry of Health of the Czech Republic, grant nr. 17-30397A. All rights reserved. This report was written with the support of the Specific University Research (nr. MUNI/A/0968/2017) provided by MEYS.

Disclosures
Mayer: Affimed: Research Funding; Eisai: Research Funding; Johnson & Johnson: Research Funding; Roche: Research Funding; Novartis: Research Funding.