A Simple and Reliable Protocol for the Preparation and Culturing of Fresh Surgically Resected Human Glioblastoma Tissue.

Glioblastoma is a heterogeneous glial cell malignancy with extremely high morbidity and mortality. Current treatment is limited and provide minimal therapeutic efficacy. Previous studies were reliant on cell lines that do not accurately reflect the heterogeneity of the glioma microenvironment. Developing reliable models of human glioblastoma is therefore essential. Direct culture of human brain tumours is often difficult and there is a limited number of protocols available. Hence, we have developed an effective method for the primary culture of human glioblastoma samples obtained during surgical resection. Culturing tumour tissue direct from human brain is advantageous in that cultures (1) more closely resemble true human disease, relative to the use of cell lines; (2) comprise a range of cellular components present in the natural tumour microenvironment; and (3) are free of added antibodies and reagents. Additionally, primary glioblastoma cultures are valuable in studies examining the effects of anti-cancer pharmaceuticals and therapeutic agents, and can be further used in live cell imaging, immunocytochemistry, flow cytometry and immunoassay experiments. Via this protocol, cells are maintained in supplemented medium at 37 °C (5% CO2) and are expected to achieve sufficient confluency within 7 days of initial culture.


Introduction
Glioblastoma is the most predominant and aggressive central nervous system malignancy, accounting for over 60% of all brain tumours in adults [1]. Current treatments are limited in range and efficacy, with patients surviving for a median of only 14 to 15 months post-diagnosis [1]. Hence, a deeper understanding of glioblastoma pathology and the mechanisms of tumour pathogenesis is pivotal for research progression. The heterogeneity of glioblastoma is reflected by the crosstalk between tumour cells, microglia and a multitude of invading immune cells and cytokines [2,3]. This presents as a hurdle to overcome when developing reliable models of glioblastoma.
Conventionally, glioblastoma research has been based on data obtained from homogeneous, murine and patient-derived cell lines. While cell lines provide a rapid and reproducible means Methods Protoc. 2020, 3, 11; doi:10.3390/mps3010011 www.mdpi.com/journal/mps of in vitro experimentation [4], they do not accurately mimic the heterogeneity of tumours in vivo.
Glioblastoma cell lines have been demonstrated to express markedly different gene expression profiles when compared to primary tumours [5,6] and are susceptible to genetic drift across passages that alter experimental reproducibility [4]. Importantly, surgically resected human glioblastoma tissue retains the molecular and cellular characteristics of the original tumour mass. These samples have also been demonstrated to express microglia, the largest immune cell infiltrates of the glioblastoma microenvironment [7,8]. Other immune cells present may include lymphocytes, neutrophils, monocytes/macrophages and myeloid-derived suppressor cells [9][10][11][12]. Cellular infiltrates serve a range of functions that disparately affect tumour growth. Hence, relative to cell lines, the culturing of samples directly from surgically resected glioblastoma more closely resembles true disease and takes into account the effect of infiltrating immune cells. Existing protocols for the culture of primary glioblastoma tissue are limited and have limited growth efficacy [13]. In light of this, we demonstrate a simple, reliable and efficient protocol for the direct culture of human glioblastoma tissue. Importantly, this protocol processes tumour samples for culturing immediately after surgical resection, which minimizes potential environmental disruptions that may significantly affect the tumour microenvironment. This method is particularly useful for drug testing in vitro, and can be used to assess the presence of various cell populations and biomarkers within the tumour microenvironment via immunocytochemistry, flow cytometry and immunoassays.

Experimental Design
Methods Protoc. 2019, 2, x FOR PEER REVIEW 2 of 7 vitro experimentation [4], they do not accurately mimic the heterogeneity of tumours in vivo.
Glioblastoma cell lines have been demonstrated to express markedly different gene expression profiles when compared to primary tumours [5,6] and are susceptible to genetic drift across passages that alter experimental reproducibility [4]. Importantly, surgically resected human glioblastoma tissue retains the molecular and cellular characteristics of the original tumour mass. These samples have also been demonstrated to express microglia, the largest immune cell infiltrates of the glioblastoma microenvironment [7,8]. Other immune cells present may include lymphocytes, neutrophils, monocytes/macrophages and myeloid-derived suppressor cells [9][10][11][12]. Cellular infiltrates serve a range of functions that disparately affect tumour growth. Hence, relative to cell lines, the culturing of samples directly from surgically resected glioblastoma more closely resembles true disease and takes into account the effect of infiltrating immune cells. Existing protocols for the culture of primary glioblastoma tissue are limited and have limited growth efficacy [13]. In light of this, we demonstrate a simple, reliable and efficient protocol for the direct culture of human glioblastoma tissue. Importantly, this protocol processes tumour samples for culturing immediately after surgical resection, which minimizes potential environmental disruptions that may significantly affect the tumour microenvironment. This method is particularly useful for drug testing in vitro, and can be used to assess the presence of various cell populations and biomarkers within the tumour microenvironment via immunocytochemistry, flow cytometry and immunoassays.

Experimental Design
Ensure ALL materials and equipment are sterile prior to usage.

Procedure
Ensure ALL experiments are completed under sterile conditions with appropriate aseptic techniques to minimize sample contamination and exposure to human tissue. 9. Transfer fragmented sample into the warm EBSS and papain solution and place in a water bath at 37 °C for 40 min. 10. Gently aspirate sample with a 10 mL pipette and transfer into a new 50 mL centrifuge tube with 3 mL culture medium. Wash in triplicate with culture medium, topping up with new culture medium after each wash.

Poly-D-Lysine Plate Coating Time for Completion
Ensure ALL experiments are completed under sterile conditions with appropriate aseptic techniques to minimize sample contamination and exposure to human tissue.

1.
Add 50 mL of autoclaved distilled water into 5 mg stock poly-D-lysine powder using a 50 mL syringe with a 0.2 µm pore syringe filter. Re-cap the stock bottle and shake lightly.

2.
Transfer one 18 mm glass coverslip per well onto 12-well cell culture plates. NOTE: A single 50 mL poly-D-lysine solution can be used to prepare approximately thirteen poly-D-lysine-coated 12-well cell culture plates.

3.
Add 300 µL of poly-D-lysine solution per well and leave at room temperature (25 • C) for 2 h. 4.
Aspirate residual poly-D-lysine solution in each well and leave in sterile conditions at room temperature for 48 h or until the wells have dried.

5.
Methods      9. Transfer fragmented sample into the warm EBSS and papain solution and place in a water bath at 37 °C for 40 min. 10. Gently aspirate sample with a 10 mL pipette and transfer into a new 50 mL centrifuge tube with 3 mL culture medium. Wash in triplicate with culture medium, topping up with new culture medium after each wash.

Sample Preparation and Tissue
CRITICAL STEP: Transfer the tumour tissue into a petri dish and gently fragment the tissue with a surgical scalpel (Figure 1).

Procedure
Ensure ALL experiments are completed under sterile conditions with appropriate aseptic techniques to minimize sample contamination and exposure to human tissue.

9.
Transfer fragmented sample into the warm EBSS and papain solution and place in a water bath at 37 • C for 40 min. 10. Gently aspirate sample with a 10 mL pipette and transfer into a new 50 mL centrifuge tube with 3 mL culture medium. Wash in triplicate with culture medium, topping up with new culture medium after each wash. 11.
Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 7 11. CRITICAL STEP: With a gentle up and down pipetting motion, use a Pasteur pipette with a rubber bulb to disassociate the cells, creating a single cell suspension. TIP: Soften the edges of the Pasteur pipette with a Bunsen burner to minimize disruption to cellular integrity. 12. Count the cells using an automated cell counter and top up accordingly with culture medium to make up 5 × 10 5 viable (live) cells/mL. Alternatively, a haemocytometer can be used. 13. Homogenize the cell solution by pipetting up and down and transfer 1 mL per well onto 12-well cell culture plates pre-coated with poly-D-lysine. Maintain cells in a humidified incubator at 37 °C with 5% CO2 for 7 days or until cells are 80% confluent. NOTE: For optimal growth, replace the culture medium every 3-4 days.

Expected Results
It is important to note that due to existing heterogeneity between each tumour, there may be a difference in proliferation rate per culture. The time taken to reach 80% confluency might vary between each culture; we have previously cultured 20 tumours using this protocol, of which 17 (85%) reached 80% confluency within a time span of 7 days.
Surgically resected human glioblastoma tissue cultured via this protocol closely reflect the cellular heterogeneity of the original tumour. In particular, glioma cells as well as microglia and other immune cells are expected to contribute to the vast majority of the cellular architecture [7,8]. In Figure 2, we show the presence of glioma cells marked by glial fibrillary acidic protein (GFAP), as well as CD11b-positive immune cell infiltrates in culture. CD11b is known to be expressed on the surface of many immune cells, including tumour-associated microglia/macrophages, monocytes, neutrophils and natural killer cells [14][15][16]. Cells in culture were fixed at 80% confluency, stained via immunocytochemistry and visualized using fluorescence confocal microscopy.

Troubleshooting
Potential issues arising and their respective troubleshooting solutions are presented in Table 1.
CRITICAL STEP: With a gentle up and down pipetting motion, use a Pasteur pipette with a rubber bulb to disassociate the cells, creating a single cell suspension. TIP: Soften the edges of the Pasteur pipette with a Bunsen burner to minimize disruption to cellular integrity. 12. Count the cells using an automated cell counter and top up accordingly with culture medium to make up 5 × 10 5 viable (live) cells/mL. Alternatively, a haemocytometer can be used. 13. Homogenize the cell solution by pipetting up and down and transfer 1 mL per well onto 12-well cell culture plates pre-coated with poly-D-lysine. Maintain cells in a humidified incubator at 37 • C with 5% CO 2 for 7 days or until cells are 80% confluent. NOTE: For optimal growth, replace the culture medium every 3-4 days.

Expected Results
It is important to note that due to existing heterogeneity between each tumour, there may be a difference in proliferation rate per culture. The time taken to reach 80% confluency might vary between each culture; we have previously cultured 20 tumours using this protocol, of which 17 (85%) reached 80% confluency within a time span of 7 days.
Surgically resected human glioblastoma tissue cultured via this protocol closely reflect the cellular heterogeneity of the original tumour. In particular, glioma cells as well as microglia and other immune cells are expected to contribute to the vast majority of the cellular architecture [7,8]. In Figure 2, we show the presence of glioma cells marked by glial fibrillary acidic protein (GFAP), as well as CD11b-positive immune cell infiltrates in culture. CD11b is known to be expressed on the surface of many immune cells, including tumour-associated microglia/macrophages, monocytes, neutrophils and natural killer cells [14][15][16]. Cells in culture were fixed at 80% confluency, stained via immunocytochemistry and visualized using fluorescence confocal microscopy.

Expected Results
It is important to note that due to existing heterogeneity between each tumour, there may be a difference in proliferation rate per culture. The time taken to reach 80% confluency might vary between each culture; we have previously cultured 20 tumours using this protocol, of which 17 (85%) reached 80% confluency within a time span of 7 days.
Surgically resected human glioblastoma tissue cultured via this protocol closely reflect the cellular heterogeneity of the original tumour. In particular, glioma cells as well as microglia and other immune cells are expected to contribute to the vast majority of the cellular architecture [7,8]. In Figure 2, we show the presence of glioma cells marked by glial fibrillary acidic protein (GFAP), as well as CD11b-positive immune cell infiltrates in culture. CD11b is known to be expressed on the surface of many immune cells, including tumour-associated microglia/macrophages, monocytes, neutrophils and natural killer cells [14][15][16]. Cells in culture were fixed at 80% confluency, stained via immunocytochemistry and visualized using fluorescence confocal microscopy.

Troubleshooting
Potential issues arising and their respective troubleshooting solutions are presented in Table 1.

Troubleshooting
Potential issues arising and their respective troubleshooting solutions are presented in Table 1. Step

11
Tissue does not easily dissociate when pipetted 1.
The tissue sample was not sufficiently fragmented in Step 8 2.
The quality/strength of the papain enzyme solution might be compromised 3.
The tissue was not left in papain solution for long enough

1.
Ensure the tissue is fragmented into smaller (<2 mm) pieces in Step 8 2.
Ensure proper storage of papain solution when not in use. Ensure that the papain solution has not expired. Increase the concentration of papain with larger samples.

3.
Leave sample in papain solution for an additional 5-10 min

13
Larger chunks of tissue are present in homogenized cell solution Tissue was not properly dissociated in Step 11

Follow the suggestions for
Step 11 (above) to ensure tissue is properly dissociated.

2.
Use a 10 mL pipette to mix cell solution and allow larger pieces of tissue to settle at the bottom. Exclude the larger pieces of tissue when transferring onto 12-well culture plates. Change culture medium after 3 days.

13
Cells are not becoming confluent 1.
The tissue was damaged by over-fragmentation in Step 8 2.
The tip of the Pasteur pipette was broken or too sharp, which damaged cellular integrity when pipetted 3.
The quality of the culture medium has been compromised 1.
Ensure tissue is fragmented gently in Step 8 2.
Smoothen the tip of the Pasteur pipette with a Bunsen burner prior to usage. Ensure tip is not damaged.

3.
Ensure proper storage of culture medium when not in use and that the medium or any component of the medium has not expired. Funding: This research has been funded by Brain Foundation (Australia).