Abstract
Background:
For studying in vitro immune responses, peripheral blood mononuclear cells (PBMCs) and its immune cell components isolated from human blood are used. In this study, we have standardized the appropriate percentage of human AB serum (HABS) for a cost-effective work layout in monocyte cultures derived from human PBMC.
Methodology:
For studying the usage of AB serum in culture, PBMCs-derived monocyte-derived macrophages (MDMs) were studied in healthy individuals (purified protein derivative (PPD) positive (n = 3), PPD negative (n = 3)) and pulmonary tuberculosis patients (PTB) (n = 3). Monocytes derived by density gradient centrifugation were adhered and differentiated into MDMs for the study. The morphology and viability of the MDMs in different percentages of AB sera (5% and 10% HABS) were assessed for optimization.
Results:
No significant difference was observed in the usage of 5% and 10% of HABS in MDMs culture in terms of morphology and viability in different groups of study subjects: PPD positive subjects (P value: 1.0000), PPD negative subjects (P value: 0.6531), and PTB (P value: 0.8195)), and 5% HABS was found to be optimum for culturing MDMs.
Conclusion:
This work is novel because to our knowledge, none of the studies have compared different percentages of human sera for evaluating their efficiency in cell culture. Since the cost of HABS is about four to five times more than that of fetal bovine serum, a study of this kind that explores the optimum usage of serum for supporting cell growth without compromising cell viability and morphology will be of much use in cell culture studies.
Introduction
Macrophages are a vital component of the innate immune system, with their circulation in lymphoid and nonlymphoid tissues all over the body. They were at first known to arise from circulating blood monocytes that constantly migrate to different tissues and differentiate into macrophages.1 Macrophages play a crucial role in host responses to intracellular bacteria-like Mycobacterium tuberculosis (MTB), wherein these cells represent the forefront of innate immune defense against bacterial invaders. Thus, to study macrophage-related immune responses invitro, monocytes derived from peripheral blood differentiated into macrophages (monocyte-derived macrophages (MDM)) are used. Since tuberculosis (TB) is an intracellular infection of macrophages leading to establishment of infection, this infection model has beenconsidered in this investigation for optimizing the human AB serum (HABS) usage in cell culture studies. In TB, MTB has the notable capacity to survive within the hostile environment of the macrophage. Macrophages have evolved a number of innate defense strategies to combat the infection against MTB.2 Alveolar macrophages are the chief site for MTB in which it resides and multiplies and then establishes the infection. MTBs are capable to infect, adapt, survive, and replicate in numerous cell types within its human host. In pulmonary infection, MTB goes through multiple environments, where alveolar macrophages constitute a key replication niche in early infection and, thus, play a central role in the tubercle bacilli lifecycle.3 In most of the studies on innate immune responses of macrophages, MDM is used as a model cell. In this study, it was aimedto optimize the usage of HABS for MDM culture studies.
Materials and Methods
Study subjects
Ethical approval from the Institute of Human Ethics Committee of the Indian Council of Medical Research (ICMR)-National JALMA Institute for Leprosy and Other Mycobacterial Diseases (NJIL&OMD), Agra (IHEC-2017) (in accordance with ICMR ethical guidelines-2006)4 was obtained before collecting the samples, and an informed consent was obtained from the study subjects before sample collection.
Normal healthy subjects
Normal healthy subjects were categorized into purified protein derivative (PPD) positive (n = 3) and negative (n = 3) subjects. This categorization was based on the reactivity of individuals to PPD, which was injected in the forearm of the participants via the intradermal route (0.1 ml of liquid containing 5TU of tuberculin PPD (Arkray, India)). The induration (swelling) that occurred to PPD reactivity (due to the presence of MTB antigens in the body indicating TB infection) was measured within 48–72 h. If the induration diameter is 10 mmor above, it was considered as PPD positive, and if it is less than 10 mm, it is considered as PPD negative. The participants were volunteers (students and staff) working in ICMR-NJIL&OMD, Agra. All the healthy individuals were clinically examined, and they had no history of tuberculosis, other mycobacterial disease, or any other infectious diseases.
None of the healthy individuals was having any symptoms of TB and leprosy, and they were free from any other infection at the time of recruitment.
Pulmonary tuberculosis patients
Treatment-naïve pulmonary tuberculosis patients (PTB) (n = 3) who were positive for acid fast bacilli by sputum smear and culture were recruited for this study from the directly observed treatment short-course (DOTS) center at ICMR-NJIL&OMD.
Exclusion criteria
In this study, healthy individuals and patients who were positive for HIV and other immunosuppressive conditions like diabetes, transplantation, etc. were excluded.
Procedure
Peripheral blood mononuclear cells (PBMCs) isolation and monocyte adherence are as follows: heparinized blood samples were collected from the study subjects (9 ml). Blood was diluted in Rosewell Park Memorial Institute Medium (RPMI) (Sigma-Aldrich, USA) with 1:1 ratio, and layering of diluted blood was done over histopaque (Sigma-Aldrich, USA). Tubes were centrifuged at 400×g for 20 min at 25°C temperature (at nine acceleration and zero deacceleration), and PBMCs (buffy white layer) were isolated after the gradient centrifugation. PBMCs were washed using the Hanks’ Balanced Salt Solution (HBSS) (Sigma-Aldrich, USA) by centrifugation at 350×g for 10 min and at 300×g and 250×g for 5 min, each at 25°C temperature (at five deacceleration). Final cell pellet was suspended in complete RPMI [RPMI media containing 5%/10% HABS (MP-Biomedicals, USA)/fetal bovine serum (FBS) 10% (ThermoFisher Scientific, USA), 0.5% glutamine (Sigma-Aldrich, USA), and 0.5% antibiotic antimycotic solution (Sigma-Aldrich, USA)]. Cell viability was checked using the trypan blue exclusion method. It was done by preparing a 1:10 ratio trypan blue (ThermoFisher Scientific, USA) staining solution, a 10 μl of cell suspension was added, and the cell count was performed using a hemocytometer (Marienfeld, Germany). After the count, 0.5 million PBMCs were seeded in each well of 96-well culture plate in duplicate, and cells were incubated at 37°C in a 5% CO2, 96% relative humidity (RH) for 2 h in complete RPMI (with 5%/10% HABS/10% FBS).
Culture of MDM
After 2 h of incubation, the supernatant with nonadherent cells was aspirated out, and adhered monocytes in the wellswere replenished with fresh RPMI media containing 5%/10% HABS/10% FBS, and glutamine and antibiotic antimycotic solution. Monocytes were incubated for 5 days at 37°C, 5% CO2, and 96% RH, and every day, the plate was observed under the inverted phase contrast microscope (Olympus, Japan) for viability and differentiation of MDM. On the 7th day, viable MDMs were observed.
Detachment of MDMs and enumeration of viability and morphology
Trypsin Ethylenediaminetetraacetic Acid (EDTA) method
Culture media from plate was aspirated out, and MDMs were rinsed with Dulbecco’s phosphate buffered saline (DPBS (Ca2+, Mg2+ free)) (ThermoFisher, USA) twice prior to adding trypsin Ethylenediaminetetraacetic Acid (EDTA). The cells were then covered with 10 or 20 µl trypsin EDTA (T/E solution) (ThermoFisher, USA). The culture plate was gently tapped to ensure complete coverage of the trypsin EDTA solution over the cells and incubated for 2–3 min at 37°C in the CO2 incubator, and then excess fluid was aspirated off from the monolayer. The cells were observed under the microscope. When the cells pulled away from each other and rounded up (typically within about 3–6 min), the plate was removed from the microscope, and the culture plate was gently tapped from several sides to promote detachment of the cells from the plate. When the majority of cells appeared to have detached quickly, an equal volume of the trypsin neutralizing solution was added to each plate. The dissociated cells were transferred to a sterile centrifuge tube and set aside while processing remaining cells in the culture plate. About 200–300 µl DPBS was added to the tissue culture plate to collect any additional cells that might have been left behind. Cells in DPBS weretransferred to the centrifuge tube containing the trypsin-EDTA-dissociated cells. Cells were centrifuged at 150×g for 3–5 min. The neutralized dissociation solution was aspirated, and the cell pellet was resuspended in 1 ml of fresh, prewarmed, complete growth medium. Morphology of the cells was then observed under the phase-contrast microscope, and the cell count was performed to check viability.
Results and Discussion
HABS is more appropriate for studying antigen-specific T cell responses in humans, but in the case of FBS, human cells respond to bovine proteins, which give unwanted background responses. Moreover, HABS has been reported to support the propagation of different human cell types such as bone marrow cells, osteoblasts, chondrocytes, and cancer cell lines.5–7 When compared to HABS, FBS carries the possibility of viral contamination.8,9 Moreover, the cost of HABS is about four to five times more than that of FBS. Hence, a study of this kind that explores the optimum usage of serum for supporting cell growth without compromising cell viability and morphology will be of much use in cell culture studies.
In this study, we have optimized the technique to culture and differentiate PBMCs from MDMs for obtaining macrophages using different concentrations of HABS (viz., 5% and 10%) and 10% FBS for a comparative analysis (Fig.1). Viability and morphology were studied in all the three healthy subjects, and it was observed that the MDM count was similar for both 5% and 10% of HABS and FBS, as mentioned in Table1. The experiment was performed thrice, and the parameters of viability and morphology were found to be more or less similar in both the percentages of HABS. Mean absorbance values were calculated for each triplicate, and the overall mean was calculated for each data point. But, this viability was comparatively lesser in FBS cultures (except for PPD negative group in which the counts were similar to HABS). In both the HABS percentages and FBS, elongated, fibroblast-like shaped cells and well-differentiated macrophages were observed in all the three healthy donors (categorized as PPD positive and negative) and in PTBs, as shown in Fig.1.
Microscopic view of viable and well-differentiated MDMs in the presence of 5% and 10% HABS along with 10% FBS (200×).
Mean cell count of MDMs in three categories of participants in different percentages of HABS and FBS
Macrophages have evolved a defense strategy against intracellular bacteria like MTB by the generation of toxic antimicrobial effectors such as nitric oxide and reactive oxygen intermediates, by stimulating microbe intoxication mechanisms by means of acidification or metal build up in the phagolysosome. Thus, macrophages play an important role in studying the innate immune system in infection.2 Since MDMs have been found to be very reliable for studying macrophage infection in humans, they have been used as a model for exploring innate immune response in the study by Dumarey etal.10 Evidence from literature suggests that HABS is suitable for MDM culture. Saghaeian-Jazi etal.11 compared the efficiency of differentiation and viability of macrophages in culture media supplemented with 10% FBS and 10% HABS (both heat inactivated). In that study, the differentiation was monitored using light microscopy for 14 days. The investigators found that differentiation initiates on day 3 along with spindled morphological changes, which were observable in media with both supplementation. On day 12, macrophages in both media had been differentiated sufficiently. However, the number of macrophages and viability were higher in media supplemented with HABS as compared to FBS.11 Similarly, in our study with 5% and 10% HABS and 10% FBS, for the MDM generation, both the percentages of human serum exhibited good viability and morphology. Literature also suggests the use of HABS over FBS in human cell culture studies. It has also been found to be useful in glioma and melanoma cancer cell line propagation.12,13 These studies also support the fact that HABS is a good candidate for supplementing cell culture media compared to FBS. In another study, the mesenchymal stromal cells cultured in medium supplemented with 10% HABS showed similar doubling times and cumulative population doubling in HABS when compared to the 10% FBS supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile. These results again strengthen the fact that the HABS is a competent FBS substitute.14
Currently, the standard supplement for cell expansion is FBS, which contains other animal-derived components; hence, its use is discouraged by regulatory agencies as it involves the risk of transmitting xenogenic infectious agents and immunization.15,16
Further disadvantages of FBS are shortage, batch-to-batch variations impacting culture reproducibility, and ethical concerns.17 Cánovas etal.18 observed that the human serum helped the growth of primary endothelial cells and cancer cell line cultures, and it seemed to be a good candidate in replacing FBS as a medium supplement in cell culture. In that study, different serum concentrations (10% and 5%) were used for cancer and endothelial cells, and this study concludes that further investigation exploring lower human serum levels in media might be needed, which may support cell cultures without compromising cell growth.18 In our study, we optimized the usage of HABS in healthy individuals and TB patients and observed that 5% HABS is appropriate for cell culture study when compared to FBS. In the case of HABS, the most rational choices for serum production are the human blood group AB because of its lack of ABO antibodies, which avoids harmful reactions in cell cultures.19,20
In conclusion, from our current investigation, the use of 5% HABS in cell culture was found to be simple, cost-effective method for preparing MDMs with morphology and viability that will be amenable for invitro functional studies of macrophages and also a lesser percentage of serum reduces background noise in the downstream applications. Further studies on immunophenotyping, cytogenetic studies, and other functional features of the MDM using different percentages of serum may help in substantiating the currently obtained results.
Footnotes
Acknowledgments
The financial support provided by ICMR, New Delhi, India, is acknowledged. The authors declare no conflict of interest.