Achieving Standardization of Cell Lines

The above challenges to the stability and standardization of cells may be tackled through a combination of attention to appropriate cell banking, quality control, and well-defined conditions and procedures for routine passage and preparation of cells.

An important first step is the establishment of a master bank, and its characterization and quality control. Subsequently, individual vials of this stock are taken to generate large "working" cell banks that are retested for critical characteristics prior to provision for routine use. This tiered system is central to assuring the long-term provision of good-quality cells (both prokaryotic and eukaryotic), and should be considered best practice for any cell culture laboratory (Fig. 9.1).

The quality control applied to each cell bank will vary depending on the application of the cells, but the core testing for all cell lines should include some means of determining viability, Mycoplasma testing, and sterility testing [7]. The suitability of the viability test method used should be established for each cell line, and the specificity and sensitivity of the Mycoplasma and sterility tests should be carefully considered, as should the use of appropriate controls and reference strains and materials. It should be borne in mind that some fastidious organisms may not be detected in standard protocols, and sterility tests may need to be extended to include microbiological growth media that will support the growth of organisms that are dependent on carbon dioxide, anaerobic or microaerophilic conditions and serum. A typical generic core testing regime for a cell bank is provided in Table 9.4.

Table 9.3 Typical examples of variation in karyotype data for Vera cells (A) and HeLa cells (B) over a period of in-vitro culture (L. Young, unpublished data; NIBSC).

Passage level of Vero cell culture

Numbers of cells with respective chromosome counta' (200 cell metaphases)

Modal no.b>

Range

(a) Vero cells

51

52

53

54

55

56

57

58

59

60

61

62

63

64

148

2

11

16

26

38

35

27

17

17

9

2

58

54-64

165

4

12

15

14

22

19

18

13

13

6

4

58

54-64

179

5

14

18

17

28

25

22

21

16

10

4

58

54-64

199

5

16

14

19

30

27

25

18

13

10

3

58

54-64

(b) HeLa cells

53/54

55/56

57/58

59/60

61/62

63

64

65

66

67

68

69/70

71/72

73/74

n + 14

0/0

0/0

0/1

1/2

5/12

17

19

23

26

24

22

20/13

6/4

3/2

66

58-74

n + 33

0/0

0/0

0/1

1/8

9/12

15

22

27

26

30

23

13/7

3/2

1/0

67

58-73

n + 76

1/5

6/8

7/9

12/14

16/17

23

24

21

16

11

6

3/1

0/0

0/0

64

53-70

n + 85

0/1

1/4

5/7

8/15

19/23

26

25

22

17

11

6

5/3

2/0

0/0

63

54-71

a' Column headers indicate the number of chromosomes per cell metaphase. b' Note that the Vero cell line has a stable modal number, and the HeLa cells a variable modal number.

Hela Str Profiling
Fig. 9.1 A cell banking scheme for cell lines.

Table 9.4 Typical quality control regimes for cell banks.

Characteristic

Types of test

Viability Growth

Morphology Species of origin

Genetic identity (i.e., authenticity) Sterility

Mycoplasma contamination Performance in respective bioassay

Trypan blue dye exclusion [57]

Recovery of cultures from frozen ampoules Plating efficiency [58]

Light microscopy

Isoenzyme analysis Karyology

STR profiling and DNA fingerprinting

Bacteriological broth and agar culture [59]

Culture [59] Hoechst stain PCR [60]

As required with use of appropriate reference materials

A Master Cell Bank provides an important reference point for any project in which cell cultures are a critical component. However, it is also important to recognize that cells are prone to variation. When individual bank vials are recovered, careful specification of the culture media, growth conditions and subculture regime are important to assist standardized results in different laboratories, and over time. In addition, for critical applications it is good practice to passage cells beyond the expected limit of use so that their continued stability of performance in the respective assays can be determined. The response of cells to pharmacological or toxic agents may vary, depending upon factors such as the cell concentration at seeding of cultures and the degree of confluency or cell density at the point of use. Accordingly, care should be taken to control and standardize the state of cells used in bioassays.

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