Analysis of the change of the cells in bile is an evolving research field in biliary pathophysiology and has potential value in diagnosis and therapy. However, laboratory studies of cell in bile across the world are scarce. Bile was collected from the clinical patients with cholangiocarcinoma (CC). To optimize the cell separation method in bile of patients with CC, we studied the factors that may affect cell vitality in bile including the dilution buffer, centrifugal force, centrifugal time, and store time and temperature. Then these factors were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable cells. The separated cells from bile were stained with CD3, CD4, CD8, CD56, TCR
Cholangiocarcinoma (CC) is a primary hepatic malignancy that arises along the intrahepatic and extrahepatic bile ducts [
In order to improve diagnosis and prognosis, sampling of bile has become a common clinical practice since the introduction of endoscopic retrograde cholangiopancreatography (ERCP) [
We collected bile from patients with CC by ERCP (
This experiment explored the key factors that affect cell vitality through the following four steps, including such factors as the dilute buffer, centrifugal force, centrifugal time, and store time and temperature. Flow chart shows the cell separation process from bile and variations in the separation procedure (Figure
Flow chart of the separation process (on the left) and variations in the separation procedure (on the right).
Pour bile samples of about 45 mL into a 50 mL tube, 200-mesh sieve; bile samples are evenly divided into three parts (15 mL), each using three different buffers for dilution (1 : 2); samples are divided into different groups including 1640 culture medium dilution group, PBS dilute group, and the diluted saline group. 45 mL bile sample was filtered through the 200-mesh sieve and evenly put into three centrifuge tubes. Then, different double dilutions were added to bile, including 1640 culture medium, PBS, and saline. The above dilution groups were centrifuged at 300 g for 10 min at 4°C for the formation of the cell layer; then, pour out on the clear liquid after mix in PBS 20 mL, with washing condition at 300 g centrifugation for 10 min at 4°C for the three dilute groups, for the formation of the cell layer, and pour out on the clear liquid after mix in PBS 100
Obtain bile samples of about 135 mL, 200-mesh sieve; 75 mL bile samples are evenly divided into five groups, using different centrifugal forces and the same centrifugal time (10 min), respectively, A (200 g), B (300 g), C (500 g), D (800 g), and E (1000 g). 60 mL bile samples are evenly divided into four groups, respectively, A (5 min), B (10 min), C (15 min), and D (20 min). Each group of 15 mL uses PBS (1 : 2) for dilution. The above groups were centrifuged at different centrifugal forces at 4°C for the formation of the cell layer; then, pour out on the clear liquid after mix in PBS 20 mL with each of the above groups, with washing condition at 300 g centrifugation for 10 min at 4°C for the five groups, for the formation of the cell layer, and pour out on the clear liquid after mix in PBS 100
Obtain bile samples of about 120 mL, 200-mesh sieve; bile samples are evenly divided into eight groups, using different store times (0, 2, 4, and 6 h) and different temperature (4°C, at room temperature), respectively, group 1 (0 h, 4°C), group 2 (0 h, at room temperature), group 3 (2 h, 4°C), group 4 (2 h, at room temperature), group 5 (4 h, 4°C), group 6 (4 h, at room temperature), group 7 (6 h, 4°C), and group 8 (6 h, at room temperature). Each group of 15 mL uses PBS (1 : 2) for dilution. The above dilution groups were centrifuged at 300 g for 10 min at 4°C for the formation of the cell layer; then, pour out on the clear liquid after mix in PBS 20 mL, with washing condition at 300 g centrifugation for10 min at 4°C for the three dilute groups, for the formation of the cell layer, and pour out on the clear liquid after mix in PBS 100
After the two washing steps, cells were resuspended in phosphate buffered saline (PBS) and stained with 1
Gating strategy to identify positive cell (CD45). (a) Events were triggered on FSC-H at a deliberately low threshold to avoid accidental exclusion of debris cells. (b) Doublet exclusion. (c) Identification of CD45-positive cells.
T-cell population was defined as lineage (CD3, CD4, CD8, CD56, TCR
Data are presented as
Choice of dilute buffer (PBS, 1640, and saline) by flow cytometry.
Choice of centrifugal force and centrifugal time by flow cytometry. Impact of centrifugal force on cell percentage (a) and centrifugal time on cell percentage (b).
Choice of store time and temperature by flow cytometry. Impact of store time on cell percentage (a), store temperature (4°C, room temperature) for 2 h (b), store temperature (4°C, room temperature) for 4 h (c), store temperature (4°C, room temperature) for 6 h (d).
The three different buffer solutions (1 : 2) were joined in bile of patients with CC. The percentage of immune cells has no statistical difference (
Under the same centrifugal time, we examined the effect of cell viability in bile with different centrifugal forces. For this evaluation, the cell vitality is affected by centrifugal force. The mean viability of centrifugal forces 200 g, 300 g, 500 g, 800 g, and 1000 g was, respectively, 29.54%, 73.69%, 62.16%, 38.19%, and 32.46%. According to statistical results, comparing centrifugal forces 200 g, 800 g, and 1000 g with 300 g, there are significant differences (
Under the same centrifugal force, we examined the effect of cell viability in bile with different centrifugal times. The mean viability of centrifugal times 5 min, 10 min, 15 min, and 20 min was, respectively, 39.86%, 73.69%, 60.10%, and 49.67%. According to statistical results, comparing centrifugal times 5 min, 15 min, and 20 min with 10 min, there are significant differences (
We compared bile stored at 4°C for 0 h, 2 h, 4 h, and 6 h after separation of immune cells from bile and then observe the change of cell vitality by flow cytometry. The mean viability of store times 0 h, 2 h, 4 h, and 6 h was, respectively, 74.55%, 63.97%, 41.22%, and 27.58%. Comparing the store times 2 h, 4 h, and 6 h with 0 h, there are significant differences (
We compared bile stored at 4°C and room temperature for 2 h, 4 h, and 6 h after separation of immune cells from bile and then observe the change of cell vitality by flow cytometry (Figures
Expression of T cells, MDSCs and neutrophils in separated cells from bile were marked by antibody. T-cell population was defined as lineage (CD3, CD4, CD8, CD56, TCR
Immune cell subsets accumulate in bile from patient with cholangiocarcinoma by flow cytometry. Proportion of immune cells and distribution of immune cells. The immune cells were stained with FITC-conjugated anti-human-CD56, APC-Cy7-conjugated anti-human-CD3, APC-conjugated anti-human-CD16, PE-conjugated anti-human-CD4, Percp-conjugated anti-human-TCR
We systematically evaluated four steps in the process of separation by measuring cell viability and absolute immune cells from the bile count. The immune cells from the bile count depended significantly on the choice of dilute buffer, centrifugation force, centrifugation time, and store time and temperature. Within the tested range, we found no effect of changing the dilute buffer, centrifugation time, and force. But storage temperature and storage time significantly influenced bile cell viability. The larger centrifugal force and longer centrifugation time make it easier to obtain cells from bile.
To reliably utilize immune cells isolated from bile, it is important to employ consistent high-quality isolation methods to obtain high-quality samples in return. The methodology defined in this paper is a well-established way to isolate immune cells from human bile. It highlights three steps in the characterization of the isolated cells by flow cytometry. The most crucial step in the isolation of immune cells is to process fresh bile as soon as possible [
The diagnosis of occult CC can be very challenging. At present, histological investigation is the standard diagnosis for CCA. However, there are some poorly defined tumor tissues which cannot be definitively diagnosed by general histopathology [
Tumors not only effectively escape immune recognition, but they also actively inhibit T-cell-mediated normal antitumor activity to promote further tumor growth and metastasis by modulating immune checkpoints, which mediate immune tolerance and inhibit the antitumor immune response [
Immune cells are essential for protection from pathogenic infections and preventing or reducing neoplastic growth [
Laboratories worldwide use different approaches when separating immune cells from bile. Here, we show that the freshly collected bile samples have crucial impact on the recovery of live cells. The different buffer solutions were joined in bile of patients with CC; experiment results show that the different dilutions have nearly no effect on the ratio of cells in bile by flow cytometry. The centrifugation force and centrifugation time have a great influence on the vitality of cells. The larger the centrifugation force or the longer the centrifugation time, the more easily cells were separated from the bile. However, the ideal centrifugal force and centrifugation time cannot be met and the cells will not be settled down. Therefore, an optimized method for isolating cells from bile in patients with cholangiocarcinoma is to centrifuge at 300g for 10 min. The shorter the time bile is stored, the stronger the viability of the cells.
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflict of interest.
Yafei Xia and Yuan Gao have contributed equally to this paper.
This work was supported by the National Natural Science Foundation of China (No. 81470263 and No. 81602496), the Natural Science Foundation of Tianjin (No. 18JCQNJC11100 and No. 18JCQNJC13400), the Tianjin Municipal Science and Technology Commission (15ZXLCSY00030), and the Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair.