A New Protocol to Obtain 80-Passage Lncap Androgen-Independent Cells

Background

In most nations, prostate cancer (PCa), one of the top 10 cancers that affect men, is the main reason why males die from cancer [1]. With the widespread use of early prostate-specific antigen (PSA) screening and magnetic resonance imaging techniques, the earlier stage of PCa can be identified. Currently, the utilize of surgery and radiation therapy has become an effective treatment for PCa [2]. In the early stage of PCa, androgen deprivation treatment (ADT) showed a produced positive therapeutic effects [3]. However, PCa patients may change from androgen-dependent to androgen-independent state after ADT 6 to 18 months of androgen deprivation therapy, giving patients with castration-resistant prostate cancer (CRPC) a worse prognosis [4]. Cancer cells are able to withstand ADT-induced tumor inhibition once PCa transitions into a hormone- independent condition. For CRPC people, there is no cure that can extend their lives [5].

In addition to androgen-dependent and androgen-independent models, there are various prostate cancer cell lines. Because the PCa cell lines PC3 and DU145 could not produce androgens, which can develop in an environment absence of hormones. Only the Lncap cell line, which secretes PSA, PsMA, and androgens while maintaining steroid hormones, still exhibits PCa androgen-dependent traits in PCa. Therefore, the prostate cancer cell line most frequently utilized is Lncap. However, it is challenging to investigate how the process of prostate cancer develops in a state of reliant progression as opposed to a condition of non-dependent androgen progression. Despite the fact that some studies offer useful details on the ways in which testosterone works, PCa is not entirely reliant on it [6-8]. However, little is known about the cellular biology of PCa’s shift from an androgen-dependent to an androgen-independent state. The absence of a perfect cell model is a significant study barrier. In a setting devoid of androgen, a PCa cell model should exhibit androgen-dependent characteristics that produce androgen receptors and change into androgen-independent characteristics.

In this protocol, we aimed to establish an androgen-independent Lncap (LncapAI) cell subsystem from an androgen-dependent Lncap (LncapAD) cell line during a castration androgen environment and to explore a more suitable method to improve the culture protocol and enhance the efficiency to establish the Lncap-AI cell line.

Materials and Reagents

Human Lncap cell line (the Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences).

RPMI Medium 1640(gibco, catalog number: 11835-030).

GlutaMAX-I (gibco, catalog number: 35050-061).

Sodium Pyruvate (gibco, catalog number: 11360-070).

Certified Foetal Bovine Serum (FBS) Charcoal Stripped (Biological Industries Pricelist, catalog number: 04-201-1A).

Penicillin-Streptomycin Liquid (Solarbio, catalog number: P1400).

Phosphate buffered saline (Solarbio, catalog number: P1020- 500).

Dulbecco’s phosphate buffered saline (Solarbio D, catalog number: 1040-500).

Trypsin Digestion solutions with EDTA (Solarbio, catalog number: T1350).

Ethanol (Keshi, catalog number: CAS 64-17-5).

Equipment

Cell culture dish (Corning Lncorporatcd, catalog number: 430167).

Pipet tips (Axygen, catalog number: T-300; T-200-Y, T-1000-B; BS-5000-TL).

Centrifuge Tube (Corning, catalog number: 430790, 430828).

Cell culture incubator: 37°C and 5% CO2.

Rainin pipet tips (1 ml) (Mettler-Toledo, catalog number: GPS-L1000).

Procedure

Grow cells in 1640 supplemented with 10% FBS, 1% Gluta- MAX-I, 1% Sodium Pyruvate, 0.1% Penicillin-Streptomycin Liquid.

Seed cells into 100 mm culture dish at a density that after 48 h of growth, they should reach ~70-80% confluence as a monolayer.

The culture dish was taken and pour the old culture medium.

Use the D-PBS to wash culture cells mixture the affluence of Fetal bovine serum by once.

Tips: D-PBS was performed by a 50 mL centrifuge tube. Pour the D-PBS without pipette to leave a bit of medium. Use hand instead of a pipette to make sure the D-PBS is trapped in the culture dish and then will dilute trypsin concentration.

Use 1 mL 0.25% trypsin (with EDTA) to digestion adherent cells.

Tips: Trypsin was used before the temperature rise to 37°C by water-bath.

Pour the trypsin after moist all the cells mildly and put the empty culture dish into an incubator for 40-50 seconds.

Use 10 mL 1640 medium to stop the digestion.

Blow and beat until the cells float.

Draw 5 mL cell suspension to a new culture dish.

Add the match drug concentration of Flutamide 5μl (Table 1) to each dish.

Table 1: Different passage to join different drug concentration.

Note*: *CS is short for concentrated solution.

*DS is short for diluted solution.

Incubator until the cells get reach ~70-80% confluence as a monolayer to cell freezing or cell passage.

Acknowledgment

This protocol was developed in the Department of Life and science, Guangxi Medical University. This work was supported by grants from the National Natural Science Foundation of China (8216110292), Guangxi Medical University Youth Science Foundation (GXMUYSF202307), First-class discipline construction-preclinical medicine (DC2300011025).

Competing Interests

The authors declare no competing interests.

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