Mastering HK-2 Cell Culture Troubleshooting Guide

Background of Cell Line Establishment

The HK-2 cell line originates from immortalized epithelial cells of the proximal tubules in the human renal cortex. These cells are sourced from normal adult kidney tissue and engineered by introducing the HPV-16 E6/E7 genes. They have been cultured for over a year in a serum-free medium. The growth of HK-2 cells relies on epidermal growth factor (EGF) and maintains a favorable proximal tubule cell (PTC) phenotype.

• Positive expression: alkaline phosphatase, γ-glutamyltransferase, leucine aminopeptidase, acid phosphatase; cytokeratin, α3β1 integrin, fibronectin.
• Negative expression: Factor VIII-related antigen, 6.19 antigen, and CALLA internal peptidease .

Common Issues Encountered During HK-2 Cell Culture

When culturing HK-2 cells, it's common to encounter various detailed issues that shouldn't be overlooked. This article aims to summarize the common problems encountered during HK-2 cell culture along with their solutions.

1. Cell Vacuolization

The issue of vacuolization needs to be considered in conjunction with the growth rate and morphology:

• If the growth rate and morphology are normal, the vacuolization may be a result of normal cell activity. They can often diminish after passaging.
• If the growth is slow and morphology is abnormal, the vacuolization may indicate autophagy. In this case, increasing the serum concentration (not exceeding 20%) or changing the serum brand can help improve the cell condition.

Figure 1

2. Cell Stringiness

The appearance of elongated cell strings can be attributed to two scenarios:

• Migration of HK2 cells: HK2 cells are not static in culture dishes and can migrate. During migration, some HK2 cells may remain in their original positions while others migrate, leaving behind elongated cell strings. In this case, the occurrence of cell stringiness is minimal, and the overall growth status of the cells is normal, requiring no specific treatment.
• Nutrient deficiency: If the majority of cells exhibit stringiness and slow growth, it may indicate nutrient deficiency. In such cases, increasing the serum concentration (not exceeding 20%) or adding 5 ng/mL EGF can be beneficial.

3.Abnormal Cell Morphology

Normal HK-2 cells exhibit a cobblestone-like growth pattern. If HK-2 cells appear crescent-shaped or irregular (as shown below), it may indicate significant changes in the culture environment, such as transitioning from serum-containing to serum-free medium. In such cases, reverting back to the original culture conditions can gradually restore the cells. However, if the cells become elongated, it may suggest nutrient deficiency.

Figure 2

4.Slow Growth

Under standard culture conditions using Pricella's complete growth media(Cat.No.: CM-0109) and a 1:3 subculture ratio, the passage cycle for HK-2 cells is approximately 3 days.

When cells exhibit slow growth and cannot be passaged within a week, it is necessary to consider whether the culture medium, particularly the serum, is suitable.

Choosing appropriate culture medium and serum, along with increasing cell seeding density, can help improve cell growth.

Selection of Culture Medium

HK-2 cells were initially established using serum-free culture conditions. Numerous studies and practical experiences have demonstrated that HK-2 cells can also grow well under conditions where MEM, DMEM, or DMEM/F12 are supplemented with 10% fetal bovine serum (FBS). However, the quality of FBS is crucial, as higher-quality FBS tends to result in better HK-2 cell status.