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. 2021 Sep 28;12(1):520.
doi: 10.1186/s13287-021-02592-3.

Novel culture media enhances mononuclear cells from patients with chronic limb-threatening ischemia to increase vasculogenesis and anti-inflammatory effect

Nuttapol Chruewkamlow  1 , Kanin Pruekprasert  2 , Phakawan Phutthakunphithak  1 , Onchira Acharayothin  1 , Tossapol Prapassaro  2 , Kiattisak Hongku  2 , Suteekhanit Hahtapornsawan  2 , Nattawut Puangpunngam  2 , Khamin Chinsakchai  2 , Chumpol Wongwanit  2 , Chanean Ruangsetakit  2 , Nuttawut Sermsathanasawadi  3
Affiliations

Novel culture media enhances mononuclear cells from patients with chronic limb-threatening ischemia to increase vasculogenesis and anti-inflammatory effect

Nuttapol Chruewkamlow et al. Stem Cell Res Ther. .

Abstract

Background: Quality and Quantity culture media (QQ culture media) was reported to enhance vasculogenesis and angiogenesis function of mononuclear cells (MNCs) from healthy volunteers. In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function.

Methods: Patients aged ≥ 18 years with CLTI caused by atherosclerosis of the lower extremities were prospectively recruited at Siriraj Hospital (Bangkok, Thailand) during July 2017-December 2018. Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood. PBMNCs were cultured in either QQ culture media or standard culture media. The number of CD34+CD133+ cells, CD206+ cells, CD4+CD25+CD127+ cells, colony formation assay, and human umbilical vein endothelial cell (HUVEC) tube formation assay in MNCs were compared between those cultured in QQ culture media and those cultured in standard culture media.

Results: Thirty-nine patients were included with a mean age of 69 ± 11 years. Diabetes mellitus was found in 25 (64%) patients. The percentage of CD34+CD133+ progenitor cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 4.91 ± 5.30% and 0.40 ± 0.46%, respectively (p < 0.0001). The percentage of CD206+ cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 19.31 ± 11.42% and 4.40 ± 2.54%, respectively (p < 0.0001). The percentage of inactive population of T regulatory cells (CD4+CD25+CD127+ cells) in MNCs cultured in standard culture media and in MNCs cultured in QQ culture media was 14.5 ± 10.68% and 1.84 ± 1.37%, respectively (p < 0.0001). The total number of colony-forming units from MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 8.86 ± 8.35 of 2 × 105 cells/dish, and 0.58 ± 1.05 of 2 × 105 cells/dish, respectively (p < 0.0001). The mean intensity of Dil-Ac-LDL uptake that incorporated into the HUVEC forming tube was 1.37 ± 0.88 in MNCs cultured in QQ culture media, and 0.78 ± 0.41 in MNCs cultured in standard culture media. (p < 0.0003).

Conclusions: MNCs from CLTI patients that were cultured in QQ culture media had a significantly higher number of CD34+CD133+ cells and anti-inflammatory cells, and higher angiogenesis-related function compared to MNCs cultured in standard culture media.

Keywords: Anti-inflammatory effect; Chronic limb-threatening ischemia; Mononuclear cells; Novel culture media; Patients; Vasculogenesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The percentages of CD34+CD133+ cells in peripheral blood mononuclear cells (PBMNCs) compared between PBMNCs cultured in Quality and Quantity culture media (QQ-media) and PBMNCs cultured in standard culture media. All experiments were performed in triplicate. Forward and side scatter plots were used to gate the entire population of mononuclear cells. The gated cells were then evaluated to identify CD3 and CD11c negative cells. The CD3 and CD11c negative cells were then gated to assess the population of CD34+CD133+ cells. Student's t-test was used for phenotypic analysis
Fig. 2
Fig. 2
The percentages of CD206+ cells (M2 macrophages) in peripheral blood mononuclear cells (PBMNCs) compared between PBMNCs cultured in Quality and Quantity culture media (QQ-media) and PBMNCs cultured in standard culture media. All experiments were performed in triplicate. Forward and side scatter plots were used to gate the entire population of mononuclear cells. The gated cells were then evaluated to identify CD3 and CD11c negative cells. The CD3 and CD11c negative cells were then gated to assess the population of CD206+ cells. Student's t-test was used for phenotypic analysis
Fig. 3
Fig. 3
The percentages of CD4+CD25+CD127+ (inactivated T regulatory cells) in peripheral blood mononuclear cells (PBMNCs) compared between PBMNCs cultured in Quality and Quantity culture media (QQ-media) and PBMNCs cultured in standard culture media. All experiments were performed in triplicate. Forward and side scatter plots were used to gate the lymphocyte populations. The gated cells were then evaluated to identify CD3+CD4+ cells. The CD3+CD4+ cells were then gated to assess the populations of CD4+CD25+and CD127+ cells. Student's t-test was used for phenotypic analysis
Fig. 4
Fig. 4
Colony formation assay. a Peripheral blood mononuclear cells (PBMNCs) cultured in standard media, bPBMNCs cultured in QQ-media, and c colony-forming unit counts per dish compared between PBMNCs cultured in QQ-media and PBMNCs cultured in standard culture media. Scale bar = 100 µm
Fig. 5
Fig. 5
The results of tube formation assay compared between peripheral blood mononuclear cells (PBMNCs) cultured in Quality and Quantity culture media (QQ-media) and PBMNCs cultured in standard media. A Bright field and Dil-Ac-LDL images compared between standard culture medium-cultured PBMNCs (PBMNC) and QQ-media-cultured PBMNCs (QQMNC). B Reference unit intensity of Dil-Ac-LDL uptake compared between PBMNCs cultured in QQ-media and PBMNCs cultured in standard culture media. Scale bar = 100 µm
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References

    1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG, et al. Inter-society consensus for the management of peripheral arterial disease (TASC II) J Vasc Surg. 2007;45(Suppl S):S5–67. doi: 10.1016/j.jvs.2006.12.037. - DOI - PubMed
    1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–967. doi: 10.1126/science.275.5302.964. - DOI - PubMed
    1. Hirsch AT, Criqui MH, Treat-Jacobson D, Regensteiner JG, Creager MA, Olin JW, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA. 2001;286(11):1317–1324. doi: 10.1001/jama.286.11.1317. - DOI - PubMed
    1. Lawall H, Bramlage P, Amann B. Treatment of peripheral arterial disease using stem and progenitor cell therapy. J Vasc Surg. 2011;53(2):445–453. doi: 10.1016/j.jvs.2010.08.060. - DOI - PubMed
    1. Adam DJ, Beard JD, Cleveland T, Bell J, Bradbury AW, Forbes JF, et al. Bypass versus angioplasty in severe ischaemia of the leg (BASIL): multicentre, randomised controlled trial. Lancet. 2005;366(9501):1925–1934. doi: 10.1016/S0140-6736(05)67704-5. - DOI - PubMed

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