An Improved Method on Isolation and Serial Passage of Chlamydia pneumoniae From Human Peripheral Blood Mononuclear Cells

Abstract

Background

Conventional method for Chlamydia pneumoniae (Cpn) isolation and propagation is technically challenging and time‐consuming. Here, we developed a method to improve the isolation and passage of Cpn collected from human peripheral blood mononuclear cells (PBMCs).

Methods

PBMCs positive with Cpn antigen (Cpn‐Ag) were isolated, then centrifuged and cultured with Hep‐2 cells after being broken. Cells were broken again and put into new Hep‐2 cells to finish totally four passages with isolated and imported Cpn. Microimmunofluorescence method was used to detect Cpn. Inclusion forming unit (IFU) number was counted for each passage. Polymerase chain reaction (PCR) method was used to detect Cpn DNA. Efficiency of different centrifugation modes was compared.

Results

Hep‐2 cells of the first and second passages were strong positive with Cpn‐Ag, the third passage was positive, and the fourth negative. Degeneration appeared in the fourth passage for isolated Cpn and third passage for imported strain. Centrifugation mode of 1,000 rpm for 2 h was the most efficient for Cpn propagation and passage.

Conclusion

This simplified method achieved efficient isolation, propagation, and passage of Cpn from PBMCs, and isolated strain was superior to imported strain on propagating ability.

INTRODUCTION

Chlamydia pneumoniae (Cpn) is an obligate, intracellular prokaryotic microorganism belonging to Chlamydia genus. It can pass through bacterial filter, and possesses its unique cyclogeny 1. Cpn is responsible for a wide range of acute and chronic human diseases, and has been associated with atherosclerosis 2, asthma 3, lung cancer 4, chronic arthritis 5, and so on. Therefore, Cpn strains become essential material of relative research. However, the conventional method of isolating and propagating Cpn with a long‐time culture in yolk sac is technically challenging and costly 6. Recent method has replaced yolk sac culture Cpn by culture in mammalian cells, but the experimental techniques of isolating and propagating Cpn in cell culture have been less than satisfactory, as many factors affect the growth of Cpn, such as the lasting time and rotation speed of centrifugation, different cell lines, methods of breaking cells, and so on. Recent reports showed that human laryngeal carcinoma (Hep‐2) cells would be the ideal cell line for Cpn isolation and propagation, but the centrifugation time and rotation speed were different in these reports 7, 8, 9, 10, 11. Consequently, the efficiency of these methods was uneven. On the other hand, isolation of Cpn from peripheral blood mononuclear cells (PBMCs) is also difficult 7. Here we compared different modes of centrifugation with moderate speed and time and established a repeatable and simplified method with high efficiency to isolate and propagate Cpn. Here we report this improved method to propagate Cpn isolated from human PBMCs.

MATERIALS AND METHODS

Cells and Reagents

PBMCs were collected from blood samples of patients. Hep‐2 cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cell‐detaching solution was 0.25% trypsin. Hydroxypropylmethyl cellulose, cell culture fluid, and DNA isolation kit were purchased from China Hua Mei Industrial Co., Ltd. (Shanghai, China). DL 2000 DNA Marker was purchased from TaKaRa Biotechnology (Dalian, China) Co., Ltd. AR‐39 Cpn strain, fluorescein isothiocyanate (FITC) conjugated Cpn‐specific antimembrane protein monoclonal antibodies TT‐401, FITC‐conjugated Chlamydia genus‐specific antilipopolysaccharide (LPS) monoclonal antibodies were from Washington Research Foundation (WRF, Seattle, WA). Polyethylene glycol (PEG) with molecular weight of 3,000 was produced by Sanyo Electric Co., Ltd., Japan. Phosphate buffered saline (PBS) used in this study contained pH value of 7.2 and phosphate concentration of 0.01 mol/l.

Isolation of PBMCs From Blood Samples

Patients’ blood samples from different batches were stored at 4°C, and plasma was aspirated out after precipitation. The blood cells left were diluted to double volume with PBS, and slowly added into centrifugation tube that contained 3 ml hydroxypropylmethyl cellulose. The blood cell suspension was then centrifuged at 1,000 rpm for 60 min. PBMCs were collected from the interphase layer and washed three times with hypotonic solutions prepared by PBS and distilled water (1:40; 12).

Survival Rate of PBMCs

A small part of PBMCs was taken from each sample and then prepared into cell suspension with concentration of 200–2,000 cell/ml. For each sample, survival rate of PBMCs was detected by trypan blue exclusion method 13. Final survival rates were acquired from averages of three separate experiments.

Lysis Rate of PBMCs With Different PEG Concentration

PBMCs were taken from each sample and mixed together to prepare cell suspension with concentration of 1 × 10⁶ cell/ml. A suspension of 20 μl was dropped onto microscope slide. The average number of unbroken cell for four microscope's visual fields was marked as A. To test lysis rates of different concentrations of PEG, 35, 30, 25, 20, 15, 10, and 5% PEG, were prepared and 20 μl of each was added onto microscope slide, respectively. We observed the immediate lysis of PBMCs and counted the average number of unbroken cell for four microscope's visual fields marked as B. The lysis rate (marked as R) was calculated as R = (A − B)/A × 100%. Final lysis rates were acquired from averages of three separate experiments. The lowest concentration with a relatively high lysis rate was chosen for use in this study.

Detection of Cpn Antigen (Cpn‐Ag) in PBMCs

Cpn‐Ag in PBMCs was detected with microimmunofluorescence (MIF) method. PBMCs suspension with concentration of 1 × 10⁶ cell/ml was prepared for use. For each PBMCs sample, a suspension of 20 μl was dropped onto microscope slide to form test spot and blank control spot. Antigen retrieval was accomplished by microwave oven irradiation at low temperature for 1 min and middle temperature for 1.5 min, and 10 μl of diluted TT‐401 (diluted 1:40 with PBS containing 0.01% Evans blue) was overlaid on each spot, which was fixed with acetone. Slides were then incubated and washed. The number of bright fluorescent regular round apple green particles was counted under fluorescence microscope. Number of these particles of blank spot subtracted from test spot gave the final number. The result was judged according to the final number 14: strong positive (≥100 particle per spot), positive (10∼100 particle per spot, containing 10), weak positive (5∼10 particle per spot, containing 5), negative (≤4 particle per spot).

Hep‐2 Cell Culture

Hep‐2 cells were grown in RPMI‐1640 with 10% fetal bovine serum in a 37°C and 5% CO₂ humidified incubator.

Isolation, Culture, Detection, and Counting of Cpn

RPMI‐1640 without fetal bovine serum was used to prepare 25% PEG, and the pH was adjusted to 8.0 by NaHCO₃. To get broken PBMCs suspension (positive with Cpn‐Ag), 0.3 ml of 25% PEG was added to equal volume PBMCs suspension in 90 s under high‐frequency vibration. Following that, 0.4 ml of broken PBMCs suspension was added to Hep‐2 cells in culture bottle. The left suspension was divided into five equal parts and added to five plate wells with Hep‐2 cells growing well. The bottle and plate were centrifuged at 1,000 rpm for 2 h at room temperature. After centrifugation, 11 ml of Cpn growth medium (4% fetal bovine serum containing gentamicin (25 μg/ml), vancomycin (25 μg/ml), amphotericin B (2.5 μg/ml), cycloheximide (0.5 μg/ml), and glucose (4.5 mg/ml)) was added into culture bottle and 0.55 ml was added to each plate well 15. On the third day of culture, Hep‐2 cells were centrifuged at 1,000 rpm for 2 h, and Cpn growth medium was changed. Hep‐2 cells were centrifuged in the same way on the fourth and fifth days. The total culture time was 7 days. Cpn‐Ag in Hep‐2 cells was then detected by MIF method. Hep‐2 cells of three wells were fixed and stained with FITC‐conjugated Chlamydia genus‐specific anti‐LPS monoclonal antibodies. Inclusion bodies of each well were counted with epifluorescence microscope 16, and the average was taken as the final number of inclusion forming unit (IFU) in one well.

Passage, Detection, and Counting of Cpn

Hep‐2 cells infected with Cpn were broken by freeze‐thaw method twice 17. The broken cell suspension (concentration of Cpn was adjusted to 10³ IFU/ml) was passed onto fresh monolayer of Hep‐2 cells in each of the five wells (0.1 ml each), and the left suspension with original concentration was added into two culture bottles with fresh monolayer of Hep‐2 cells. Bottles and plate were centrifuged at 1,000 rpm for 2 h to prompt the transfection of Cpn into Hep‐2 cells. Hep‐2 cells were centrifuged in the same way on the third, fourth, and fifth day of incubation. The total culture time of first passage was 7 days. The second to fourth passages were operated in this way. Detection of Cpn‐Ag and counting of IFU for each passage were performed as previously described.

Detection of Cpn DNA by PCR

DNA was extracted from Hep‐2 cells in the second passage of isolated Cpn strain, using a DNA isolation kit (China Hua Mei Industrial Co., Ltd.) according to the manufacturer's instructions. The primers were designed from GenBank gene sequences using the computer program Primer Premier 5.0 and Oligo 5.0. The sequence of the forward primer is HL‐1: 5′‐GTT GTT CAT GAA GGC CTA CT‐3′ and the reverse primer is HR‐1: 5′‐TGC ATA ACC TAC GGT GTG TT‐3′. The primers directed the amplification of 437 bp DNA fragment of Cpn under such polymerase chain reaction (PCR) condition—denaturing: 94°C for 5 min; amplification cycles: 35 cycles with 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min; extension: 72°C for 7 min. PBS was used as the negative control and the DNA extracted from the Cpn‐Ag of AR‐39 strain was used as positive control. The size of the PCR product was determined by agarose gel electrophoresis with DL 2000 DNA Marker.

Efficiency Comparison of Different Centrifugation Modes

According to previous reports, different centrifugation modes of 1,700 rpm for 1 h 7, 1,200 rpm for 1 h 9, 900 rpm for 1 h 10, and 700 rpm for 30 min 8 were applied, respectively, in the whole experimental procedure containing the collection of PBMCs and isolation, culture of Cpn. All other experimental conditions were the same. IFU counts of the second to fourth passages with different centrifugation modes were all obtained in the same way.

Passage of Imported Cpn

The imported AR‐39 Cpn strains (store concentration was 10⁶ IFU/ml) were diluted to suspension of 10³ IFU/ml with Cpn growth medium. 0.1 ml of Cpn suspension was passed onto fresh monolayer of Hep‐2 cells in each of the five wells. Then we conducted the first to fourth passages and counting of Cpn in the same way as isolated Cpn strains.

RESULTS

Survival Rate of PBMCs Detected by Trypan Blue Exclusion

We kept the blood samples at 4°C for various time periods and counted the survival rate of PBMCs. We found that samples stored for 1 day had a survival rate of 93.1%, 2 days 70.9%, and 3 days 46.3%. Survival rates of PBMCs for samples stored more than 3 days were lower than 20%.

Lysis Rate of PBMCs

We compared PBMCs lysis rates with different concentrations of PEG, and found that 35, 30, and 25% PEG, were similar in lysis rate (92.2, 92.0, and 90.4%, respectively), while rates of other concentrations were lower than 80%. Therefore, 25% PEG was the lowest concentration with a relatively high lysis rate.

Result of Cpn Isolation, Culture, and Detection

After 7‐day culture with broken PBMCs, we found that the Cpn‐Ag of Hep‐2 cells was strong positive, Hep‐2 cells of the first and second passages were all strong positive (Fig. 1), the third passage positive, and the fourth weak positive.

Figure 1.

MIF pictures confirmed Cpn‐Ag particles inside Hep‐2 cells for isolated Cpn. (a) Many bright fluorescent regular round apple green particles were observed in Hep‐2 cells cultured for 7 days in the first passage. (b) Well‐distributed apple green particles were also observed in Hep‐2 cells cultured for 7 days in the second passage. Magnification, ×400.

Detection of Cpn DNA in Hep‐2 Cells of the Second Passage

In the DNA electrophoresis gel picture, a band between 400 and 500 bp was found as the PCR product of Cpn DNA extracted from Hep‐2 cells cultured for 7 days in the second passage, and the actual size of the Cpn DNA fragment is 437 bp. It confirmed the existence of Cpn in the second passage (Fig. 2).

Figure 2.

PCR result confirmed the existence of Cpn in Hep‐2 cells. (a) Negative control. (b) Amplification product of DNA extracted from AR‐39 Cpn‐Ag. (c) Amplification product of DNA extracted from Hep‐2 cells of the second passage.

IFU Counts for Different Centrifugation Modes

IFU count of Cpn in cells after 1,700 rpm for 1 h centrifugation was less than the count of 1,200 rpm for 1 h centrifugation, and the count of 700 rpm for 30 min centrifugation was the least. Centrifugation with 1,000 rpm for 2 h yielded the highest IFU count, showing that IFU counts did not increase along with speed of centrifugation. Moreover, decrease of propagation ability appeared in the fourth passage for all centrifugation modes (Table 1).

Table 1.

IFU Counts From Four Passages of Different Centrifugation Modes

	IFU counts
Centrifugation	Original	First	Second	Third	Fourth
mode	counts	passage	passage	passage	passage
1,700 rpm for 1 h	100	5,980	2,074	485	82
1,200 rpm for 1 h	100	6,712	1,981	596	90
1,000 rpm for 2 h	100	6,983	2,389	987	91
900 rpm for 1 h	100	4,560	1,697	364	67
700 rpm for 30 min	100	1,271	456	153	39

Counts are average of two separate experiments. The IFUs of these passages were all generated from 100 initial IFUs. The 100 initial IFUs of the second to the fourth passages were derived from the prior passage.

IFU Counts of Four Passages for Both Imported and Isolated Cpn

For isolated Cpn strains, IFU quantity of the fourth passage was less than original quantity, so the decrease of propagation ability appeared in the fourth passage. The imported Cpn presented this decrease in its fourth passage (Table 2).

Table 2.

IFU Counts From Four Passages of Isolated and Imported Cpn Strains

	IFU counts
Cpn	Original	First	Second	Third	Fourth
origin	IFU counts	passage	passage	passage	passage
Isolated	100	6,983	2,389	987	91
Imported	100	3,126	892	87	75

Counts are average of two separate experiments. The IFUs of these passages were all generated from 100 initial IFUs. The 100 initial IFUs of the second to the fourth passages were derived from the prior passage.

DISCUSSION

Human granulocytes are able to internalize Chlamydia in an opsonin‐independent manner. Significantly, phagocytosed Cpn were not killed, and the ingested bacteria survived and multiplied within polymorphonuclear neutrophils 18. Moreover, Cpn can extend the lifespan of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection 19. Based on this phenomenon, we collected PBMCs infected by Cpn as the source of Cpn. If we can transfer Cpn from PBMCs into Hep‐2 cells and propagate, we can avoid the expensive procedures of importing Cpn strains from certain research institutes. In our research, we broke PBMCs with 25% PEG, and transferred Cpn released from PBMCs into Hep‐2 cells successfully. Through trypan blue exclusion method, we observed that blood samples stored for three days still had a survival rate of 46.3% for PBMCs. Therefore, blood samples infected with Cpn and stored for less than three days can be the reliable and low‐cost source of Cpn strains.

For improvement of isolation of Cpn, results with different cell lines have been compared and Hep‐2 cell was found more suitable for Cpn isolation 9. By freeze‐thaw method, Hep‐2 cells could be broken completely in our study, thereby released Cpn strains survived in them. By the way of proper centrifugation, Cpn strains could be transferred into fresh Hep‐2 cells successfully (Fig. 1). But different centrifugation modes had different efficiency. According to data in Table 1, we found that centrifugation mode of 1,000 rpm for 2 h was the most efficient in all these chosen modes, and also the most suitable for Cpn strains to transfer into Hep‐2 cells. Thus, speed and lasting time of centrifugation were key factors to influence the propagating ability of Cpn. In summary, this method of propagation was simple, convenient, and efficient for common laboratories with routine equipments to conduct the whole process.

As yet, the detection of Cpn still lacks gold standard. The MIF method and PCR method are generally used, possessing ideal sensibility and specificity 20. In our study, TT‐401 used in MIF method were Cpn‐specific antibodies that avoided the detection contamination from other species of Chlamydia. After 7‐day culture with broken PBMCs, the Cpn‐Ag of Hep‐2 cells was strong positive, Hep‐2 cells of the first and second passages were all strong positive, the third passage positive, and the fourth passage weak positive. We also used PCR method to confirm Cpn DNA in Hep‐2 cells of the second passage, as to further demonstrate the existence of Cpn in the second passage. All these demonstrated that the isolation and propagation of Cpn were successful.

In this study, we also used FITC‐conjugated Chlamydia genus‐specific anti‐LPS monoclonal antibodies to detect the passage ability of isolated and imported strains. We found that the decrease of propagation ability appeared in the fourth passage for isolated strains, while the decrease appeared in the third passage for imported strains (Table 1). Thus, isolated strain may be superior to imported strain on this point. Cpn strains can be cultured in large quantity if this regression phenomenon of propagation ability can be delayed, but it remains challenging at present 21, 22.

In conclusion, we established a simplified and feasible method to isolate and propagate Cpn. Although decrease of propagation ability appeared in the fourth passage for isolated strains, it would not be a technical barrier as it is relatively convenient to obtain fresh Cpn strains from PBMCs of clinical blood samples.