1. Culturing Normal Human Cells

Table of Contents

• 1.1 Safety Considerations
• 1.2 Handling of PromoCell Normal Human Cells
• 1.3 Handling of PromoCell Culture Media
• 1.4 Subculture of Normal Human Cells with PromoCell DetachKit
• 1.5 Cryopreservation of Normal Human Cells with PromoCell CryoSFM
• 1.6 Counting the Cells
• 1.7 Biological Growth Characteristics
• 1.8 Transient Transfection of Normal Human Cells with Plasmid DNA (Lipofection Method)
• 1.9 Trouble Shooting Guide for Culturing Normal Human Cells
  
  

1. Culturing Normal Human Cells

1.1 Safety Considerations

PromoCell products should only be handled by laboratory staff instructed in good laboratory practice and discipline. All work with cell cultures and human material should be performed under proper containment. Guidelines for handling products of human origin should be adhered to at all times:

• Always wear a laboratory coat
• Ensure that approved protective clothing such as gloves and safety glasses are available and are being used
• Do not eat, drink or smoke
• Do not pipette by mouth
• Label all material clearly so that everybody can understand its nature
• Keep the laboratory area clean
• Wash your hands after performing each procedure and before leaving the laboratory area
• Any material of animal and human origin represents a potential biohazard. Although the human cells have been tested for and found negative for HIV-1 as well as Hepatitis B and C by PCR technology, take extra precautions to avoid becoming infected.

Special precautions must be taken when working with hazardous biological materials at liquid nitrogen temperatures:

• Always wear insulated gloves, safety glasses and a laboratory coat to protect the skin from exposure
• Always thaw and open vials containing hazardous material inside a biological safety cabinet
• Be prepared for vials to explode or leak

PromoCell products are for research use only and are not approved for human or veterinary use or for therapeutic or diagnostic procedures.

1.2 Handling of PromoCell Normal Human Cells

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1.3 Handling of PromoCell Culture Media

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Medium requirement for commercially available plasticware:

Culture dishes	35 mm	60 mm	100 mm	150 mm
Growth area	9.6 cm²	20.4 cm²	57 cm²	143 cm²
Medium requirement	2mL	4mL	11mL	25mL
Multiwell plates	6-well	12-well	24-well	48-well
Growth area/well	9.6 cm²	3.5 cm²	1.9 cm²	1.1 cm²
Medium requirement/well	3.0 mL	2.0 mL	1.0 mL	0.5 mL
Culture flasks	T-25	T-75	T-150
Growth area	25 cm²	75 cm²	150 cm²
Medium requirement	5 mL	15 mL	30 mL

1.4 Subculture of Normal Human Cells with PromoCell DetachKit

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1.5 Cryopreservation of Normal Human Cells with PromoCell CryoSFM

In a serum-free cell culture system, it is essential to cryopreserve cells also in a serum-free freezing medium and not in a serum-containing medium. When using an ordinary serum-containing freezing medium, the serum affects the cell culture system over a long period. This influence can only be removed by dilution through many subsequent passages. PromoCell Cryo SFM is a formula for cryopreservation of animal and human cells containing no serum but, instead, DMSO, methylcellulose and other cryoprotectant components. After cryopreservation and thawing, a very high percentage of viable cells is obtained. Cells cryopreserved in CryoSFM show also excellent attachment capacity as well as growth performance after thawing.

• Detach adherent cells with trypsin/EDTA (see handling instruction "Subculture of Normal Human Cells with PromoCell DetachKit")
  
• Neutralize trypsin with trypsin inhibitor solution
  
• Sediment cells by centrifugation at low speed (220xg) for 3-5 minutes and remove the trypsin inhibitor solution
  
• Resuspend the cells in cold (4°C) CryoSFM at a concentration of 1-4 million cells/mL.
  
• Freeze the cells gradually(1° C/minute) to -196°C and store them immediately in liquid nitrogen.

1.6 Counting the Cells

The growth rate of most adherent cells depends on the initial seeding density. Cultures seeded at low seeding density grow very slowly, whereas cultures seeded at high seeding density must be subcultured too often which results in a shortened lifespan. PromoCell therefore recommends that the cells are counted before each subculture procedure.

1.6.A. Hemocytometer Cell Counts and Viability Test

To ensure that a cell culture is growing exponentially, it is useful to know the percentage of viability and the percentage of dead cells and, hence, the growth phase of the cells. When a cell suspension is diluted with trypan blue solution, viable cells remain small, round and refractive, while non-viable cells become larger, swollen and dark blue. Both the total count of cells/mL and the percentage of viable cells can thus be determined.

1.6.A.1. Preparations

• Dissolve 0.4 g trypan blue in 100 mL physiological saline. Pass the solution through a 0.22 µm filter to remove any debris
  
  Caution: Trypan blue is harmful if ingested or inhaled. It is irritating to the eyes, harmful by skin contact and has been found to cause cancer in laboratory animals. Appropriate precautions should be taken when handling trypan blue
  
  
• Carefully clean all surfaces of the hemocytometer and coverslips with 70% Ethanol
• Moisten the edges of the coverslip or breathe on the chamber to provide moisture before placing the coverslip onto the counting area
• Gently move the coverslip back and forth over the chamber until Newton rings (rainbow-like interference patterns) appear, indicating that the coverslip is in the correct position to allow accurate counting
• Use a light microscope with magnifications of x40 to x100
• Prepare a cell suspension

1.6.A.2. Cell Counting

• Mix the cell suspension gently and add an aliquot to the trypan blue solution to obtain a 2-, 4- or 10-fold dilution in trypan blue, depending on the concentration of the cell suspension
• Draw a sample into a Pasteur pipette after mixing thoroughly and allow the tip of the pipette to rest at the junction between the counting chamber and the coverslip. Capillary forces will draw the fluid into the chamber. Fill both halves of the chamber to allow counting in duplicate
• Place the chamber under the microscope, focus on the counting chamber
• Allow the cell suspension to settle for at least 10 seconds
• Count the number of cells (stained and unstained separately) in the four corner sections of the chamber. As a rule, the cells in the left-hand and top markings of the grid should be included in each square, and those in the right-hand and bottom markings should be excluded

Calculation of Cell Counts

Number of viable cells: N x B x 10⁴ = viable cells/mL, where

• N is the average count of unstained cells per square of the four corner squares counted
• B is the dilution factor in trypan blue

Multiply the number of cells/mL by the volume of cell suspension to obtain the total number of viable cells

Number of dead cells: D x B x 10⁴ = dead cells/mL, where

• D is the average count of stained cells per square of the four corner squares counted
• B is the dilution factor in trypan blue

Multiply the number of cells/mL by the volume of cell suspension to obtain the total number of dead cells

Total cell count = viable cell count + dead cell count

% viability = viable cell count / total cell count x 100%

1.6.B. Electronic Cell Counts

Quick, accurate, reproducible counts of cultured cells can be obtained by using electronic cell-counting technology.

Principle of electronic cell counting: Cells are suspended in an isotonic electrolyte solution and drawn through a measuring capillary to which an electric current is applied via two platinum electrodes. Each particle present in the measuring capillary leads to an electric pulse. Modern electronic cell counters allow to determine both the cell number and the range of cell size.

Each cell type creates a characteristic range of cell size which allows to quantify the amount of cell debris, dead cells, viable cells and cell aggregates in the sample. The distinction between viable and dead cells is based on the fact that dead cells or damaged cells have a porous cell membrane and are, therefore, only detected according to the size of their nuclei.

1.7 Biological Growth Characteristics

Normal human cell cultures can have different growth characteristics depending, for example, on donor, donor's age and origin of tissue. Strains of normal human diploid cells have the common characteristic of evolving through serial divisions to a common endpoint at which the probability of completing the cell cycle becomes very low (senescence).

When recording the phase of a cell population, age should be expressed as population doubling level (PDL), rather than passage number. The term "passage" is often used in laboratories to indicate the age of cell cultures; however, it indicates only the number of trypsinization steps performed during the culture period. It is therefore inadequate for describing the age of a culture because trypsinization can be performed at different split ratios.

The growth of cells corresponds to a geometric progression 2⁰, 2¹, 2², 2³ ... 2ⁿ because cells are multiplying through cell division. The generation number, n, is called population doubling and is calculated according to the following equation:

n = (log Y - log X) / 0.301, where

• Y is the final cell count and
• X is the inoculation cell count

The generation time, T_G, is defined as the time required to perform one doubling and is calculated according to the following equation:

T_G = (log 2 x T) / (log Y - log X), where

• T is the time in culture (hours or days) at the time of cell counting,
• Y is the final cell count and
• X is the inoculation cell count

1.8 Transient Transfection of Normal Human Cells with Plasmid DNA (Lipofection Method)

By providing the following short protocol, PromoCell would like to offer scientists a starting point for their transfection experiments. However, each customer should optimize his/her own transfection system because it is our experience that there is no general transfection method available.

Transfection Reagents

Liposome transfection reagents are able to bind DNA and RNA because of their positive charge. The liposome/nucleic acid complex fuses with the cell membrane, and the genes introduced will be expressed in the nucleus or cytoplasm.

Ratio of Plasmid DNA to Transfection Reagent

Ratios of 1:2.5, 1:5 and 1:7.5 (DNA to Transfection Reagent) have yielded good transfection efficiencies in normal human cells. Detailed information about the recommended ratio is listed in the product information sheet for the transfection reagent.

DNA Quality

High-quality plasmid DNA is essential for achieving high-efficiency transfections. CsCl-banded DNA is often used, but this is a time-consuming method. Other Kits for plasmid purification methods are available from different suppliers.

Cell Cultures and Seeding Density

It is recommended that normal human cells are transfected at an early population doubling level. Transfect cells at 70-90% confluence in the mid-log phase of the culture. Our experience at PromoCell is that growing normal human cells to subconfluence instead of seeding them 24 hours prior to transfection results in higher transfection efficiencies. Use 6-well or 24-well culture plates for transfection experiments so that enough cells are available for reporter gene analysis.

Do not use serum and antibiotics during the transfection procedure.

Short Transfection Protocol for Normal Human Cells in 6-Well Plates

• Seed cells at a density of 5,000 to 20,000 per cm² in the corresponding PromoCell growth medium and change the medium every 2-3 days until the cells reach 70-90% confluence.
• Follow the instructions of the liposome transfection reagent supplier for preparing the liposome/DNA complex.
• Wash cells twice with the corresponding PromoCell basal medium, prf (phenol red-free), to reduce residual serum content.
• Dilute the liposome/DNA complex to a total volume of 1 mL/well with the corresponding PromoCell basal medium, prf (phenol red-free), add the diluted complex to the cells and incubate the culture for 3-5 hours at 37°C and 5% CO₂.
• After incubation, change medium to the corresponding PromoCell growth medium for optimal growth of normal human cells after transfection.
• Assay cell extracts for reporter gene activity after 12 to 48 hours in a time experiment.

1.9 Trouble Shooting Guide for Culturing Normal Human Cells

When culturing normal human cells, one needs considerable experience to achieve a high yield of viable cells during the culturing procedure. Many of the more common errors may be avoided if the instruction sheet is read and fully understood. Use the following table to determine the possible reasons for unsatisfactory results and to improve future cultures.