Highveld Biological

SYNTHETIC CULTURE MEDIA FOR MAMMALIAN CELLS

THE RECIPE
The composition of all standard powdered and liquid media for the culture of eucariotic cells, follow precisely the formulations published in the scientific literature. Media made to customer's specification adhere strictly to the recipes provided and are manufactured in close discussion with the customer so that the end product suits his needs. The record of every medium made by Highveld Biological, since the start of the company, can be retrieved and repeated as long as the control number (CN... ) is cited.

A system of cross-checking a prepared recipes is designed to eliminate possible errors in every batch preparation.
RAW MATERIALS
The only criterion for the purchase of raw materials and components is their persistent high quality and continuous availability. The lot number of every raw product is recorded and can be traced to its source.

Some standard powdered culture media are manufactured for Highveld Biological and held in large batches in a controlled environment. Large volumes of custom-made media for industrial purpose are also available.
PRODUCTION OF POWDERED MEDIA
The basis for the manufacture of each batch is the prepared recipe, which includes the sequence in which ingredients have to be added and lists precautions in dealing with sensitive chemicals. Salts that contain crystal water, are dried under vacuum at low temperature with specially designed equipment and fine milling of the powder mixture makes for good solubility of the product. The appropriate weight per litre of medium is given on each container of powdered medium.

QUALITY ASSURANCE
From each batch of dry powdered medium, a sample of liquid medium is made and tested for physical and biological performance:
  1. Osmolarity is determined using freezing point depression after the osmometer is calibrated against a standard solution.


  2. pH is measured to ensure compliance of the product with product specification after the pH meter is calibrated against standard solutions.


  3. The biological performance of the medium is tested by cell growth studies over 10 days of one attached and one suspended line and a single-cell cloning assay. Possible cytotoxicity will become obvious by optical evaluation of 5 to 6 different cell lines under conditions of low cell density re-growth.


  4. Endotoxin tests are carried out using the LAL methodology.
STORAGE OF POWDER MEDIA
As powdered media are very hygroscopic, it is essential that they are stored in cool, dry place. A container, once opened, should be kept well wrapped in plastic and desiccated.
MANUFACTURE OF LIQUID MEDIA
The quality of the water is the most important aspect of liquid media preparation. Highveld Biological is using a double reverse osmosis plant that produces water of conductivity of less than 1 micro siemens/cm2. The water is stored in a stainless steel tank where it is continuously circulated and before use, passed through a pyrogen absorbing filter.

Liquid medium is made in 5 litre to 200 litre batches. The preparation of recipes, weighing and mixing is carried out according to a production protocol. Osmolarity and pH are checked and adjusted if necessary to ensure that the parameters of the final product are within the appropriate range of values.

Aseptic filtration and filling into final containers is carried out in clean rooms under laminar flow hoods, which are equipped with HEPA filters and run under positive pressure. The laminar flow hoods are regularly monitored for adherence to internationally accepted standards.

The liquid medium is filtered through validated cone filters of absolute 0.22µ pore size using 5 kPa positive pressure.

At regular intervals during the filtration process, samples are taken and labeled for sterility control, in all 5% of every production batch. At the end of each filtration run, 500ml of nutrient broth is pumped through the system and included in the tests.

Filled bottles are fitted with tamper-proof viscose rings, labeled with preliminary pull-off stickers and stored at 4°C in a quarantine area until the sterility and other tests are finalized. If the batch of medium meets our final specification, the bottles are labeled, packed, stored in a specified space - ready for dispatch.
QUALITY CONTROL OF LIQUID MEDIA
The 5% portion of filtered medium set aside for sterility control is incubated at 37°C for 5 days and thereafter 100µl from each container inoculated into 5ml nutrient broth and 5ml Sabouraud medium. The tubes are read 3 times over 10 days; the batch is only cleared for release when all tubes remained clear.
STORAGE OF LIQUID MEDIA
Liquid media should be stored at 4°C and exposed to light (in particular UV light) as little as possible. Media should be warmed to room temperature before use - water baths are to be avoided because they often lead to high levels of background contamination.

It is not advisable to freeze liquid media - insoluble complexes may develop.
CUSTOM-MADE MEDIA
Enquiries are welcome for custom-made liquid or dry powdered media used for specific applications. These may be media lacking in one or other ingredient and used for radio-isotope studies or special formulations cited in the scientific literature. It is essential that the customer furnish a complete and signed recipe so that there is an accepted basis for our production.

The minimum amount of custom-made medium we can supply is 10L powder or 20 x 500 ml liquid medium.

As all custom-made media and solutions are quality controlled in terms of physical parameters and biological performance, the minimum time for manufacture is 3 to 4 weeks. Highveld Biological will be in contact with the customer with respect to enquiries and suggestions as far as these special media are concerned.
HEPES BUFFERED MEDIA
HEPES buffered media help to stabilize pH fluctuations of fastidious cell cultures - the organic compound is one of several hydrogen ion buffers which act in the range of 7,2 to 7.6. HEPES is to be used together with sodium bicarbonate as the latter is an essential nutrient for cells in culture. The addition of HEPES increases the osmolarity of the medium which may necessitate the lowering of the NaCl content in the basic formulation or the addition of a calculated amount of water.
SHELVE LIFE OF LIQUID MEDIA
Most tissue culture media are stable for at least 18 months if kept between 4°C and 8°C. The only components, which decay over time are antibiotics and glutamine.

Some laboratories with experienced staff manage to culture cells without the addition of antibiotics. In most cases, however, one or other kind of antibiotic is used, either as a matter of routine or intermittently.

Unless specifically excluded, powdered tissue culture media contain L-glutamine that is stable in dry form. Some commercial liquid media however do not contain glutamine and require addition before use. Highveld Biological's liquid media are manufactured to include glutamine because we have sufficient control over our distribution and cold chain. If a customer is not sure how long some medium has been on his shelf, we advise the addition of 5 ml standard solution of L-glutamine (100mM) to 500ml medium.

Note: it is not advisable to freeze liquid media - insoluble complexes may develop.
SERUM SAVER MEDIA
Highveld Biological has developed a series of serum-saver media that consist of standard media with added growth factors and 01% Dextran sulfate. The latter is included in order to increase the viscosity of the media. In this environment many cell types grow satisfactorily at serum levels of only 1 to 3%.
SERUM-FREE MEDIA
The use of serum-free medium in cell culture technology is becoming more frequent as new formulations are published. The great advantage, of course, lies in the fact that cells which grow under chemically defined conditions are much more available for studies of normal/abnormal functions - after all, fetal calf serum is not the milieu of most cells and tissues. Although some serum-free media are published in the scientific literature, most commercial suppliers consider the formulations "proprietary" and so deprive the user of the full knowledge of the chemical formulation.

Highveld Biological offers several serum-free media with disclosure and discussion of the formula. Through our expertise in the production of small amounts of custom-made media, we are able to assist in the development of interesting biological work using cells that grow and perform in defined medium without the interference of undefined serum factors

USEFUL INFORMATION

MANUFACTURE OF LIQUID MEDIA FROM DRY POWDER
  1. Use the entire content of a container or alternatively weigh the appropriate amount of medium.


  2. Dissolve in chemically pure water at ambient temperature (see below) by adding 75% of the intended volume. Do not heat the water.


  3. Stir slowly by hand or on a magnetic stirrer for 10 minutes.


  4. Weigh the amount of sodium bicarbonate (NaHCO3) per litre as stated on the container and dissolve in the remaining 25% of the water.


  5. Add the bicarbonate solution to the medium and mix well; If required add antibiotics, HEPES and/or fetal calf serum.

  6. Adjust the pH of the medium to 0.2 or 0.3 below the desired value with 1N NaOH or 1N HCL because the pH usually rises during the process of filtration.

  7. Certain media require adjustment of the osmolarity, in particular when HEPES has been added. The desired osmolarity for mammalian cells is 270 to 290 mosmol/kg H²O, but other values may be required. The adjustment is done as follows:


    8. Actual osmo - Desired osmo
    Desired osmolarity = Desired osmo x volume

  8. Filter the medium as soon as possible through a sterile 0.22µ disc or cone filter using positive pressure if possible.


  9. Close the containers tightly, label and store at 4°C.

For quality control of sterility, remove 5% of the medium during the filter process (including the first and last filtrate), label and incubate for 5 days at 37°C. Then inoculate 5ml of bacterial broth medium and 5ml of Sabouraud medium with 100µl test medium. Incubate the samples at 37°C and room temperature respectively for 14 days; record the results.

PREPARATION OF SINGLE STRENGHT MEDIUM FROM 10X (OR 5X) LIQUID CONCENTRATES
  1. This procedure must be carried out under aseptic conditions using sterile components to achieve an acceptable final single-strength solution.


  2. To make 1l of single-strength medium, fill an adequate glass container with approximately 700ml of pure, sterile biotechnology water.


  3. Aseptically add 100ml of 10x concentrated medium (or 200ml of 5x concentrated medium) and mix gently.


  4. Aseptically add 10 ml of 100x concentrated L-glutamine solution (100mM) and the desired amount of 7.5% sodium bicarbonate solution (see table below). If required, add 10 ml of a 100x concentrated solution of penicillin/streptomycin or penicillin/streptomycin/fungizone.


  5. Adjust the pH to 7.1 to 7.3 by adding sterile 1N HCL or 1N NaOH solution aseptically - take small samples and determine the pH values using a pH meter.


  6. Store all media at 4°C to 8°C.


  7. For sterility quality control remove samples and incubate neat and on broth and Saboraud medium


MEDIA FOR THE CULTIVATION OF INSECT CELLS
Insect cells are increasingly used in applied research and manufacturing biotechnology, mainly because of the successful application of the Baculo virus expression vector system (BEVS).

Several insect culture media are available from Highveld Biological as ready-to-use liquid products. The media are tested on Spodoptera frugiperda Sf-9 cells and Drosophila melanogaster Dros-2 cells.

PRODUCTS FOR CELL CULTURE AVAILABLE FROM HIGHVELD BIOLOGICAL

Media
L19 Alpha MEM L07+ Dutch RPMI (w Hepes)
P19 Alpha MEM powder P07+ Dutch RPMI (w Hepes) powder
LXX-AT Attachment medium L05 Eagle's MEM (Earle's / Hanks)
L01 Basal Medium Eagle C05 Eagle's MEM (10xconc)
L01-AD Basal Medium Eagle Add C05A Eagle's MEM (5xconc)
C01 Basal Medium Eagle (10xconc) P05 Eagle's MEM (E/H) powder
P01 Basal Medium Eagle powder C12 Fischer's Medium (10xconc)
220 BME amino acids (50xconc) L12 Fischer's Medium
L14+ BSK Medium P12 Fischer's Medium powder
L14 CRML-1066 Medium L17 Glasgow Medium (w/o TPB)
P14 CRML-1066 Medium powder C17 Glasgow Medium (5xconc)
P-cm Custom-made powder medium P17 Glasgow Medium powder
LXX-DS Double strength medium L16 Grace's Medium
L02 Dulbecco's MEM L08 Ham's F10 Medium
L02/08 DMEM / Ham's F10 C08 Ham's F10 Medium (10xconc)
L02/03 DMEM / Ham's F12 L08-add Ham's F10 Add
C02 Dulbecco's MEM (5xconc) P08 Ham's F10 Medium powder
P02 Dulbecco's MEM powder
L03 Ham's F12 Medium L06 M199 Medium
C03 Ham's F12 Medium (10xconc) C06 M199 Medium (10xconc)
P03 Ham's F12 Medium powder P06 M199 Medium powder
L13 Iscove's Medium (w Hepes) L18 MCDB Medium
P13 Iscove's Medium powder L04 McCoy 5A Medium
L21 Joklik's Suspension MEM C04 McCoy 5A Medium (10xconc)
C21 Joklik's Suspension MEM (5xconc) P04 McCoy 5A Medium powder
P21 Joklik's Suspension MEM powder L23 NYSF-404 Medium
L11 KSLMS Medium serum free L07 RPMI 1640 Medium
L09 Leibowitz L15 Medium P07 RPMI 1640 powder
P09 Leibowitz L15 Medium powder L22 Schneider's Drosophila Medium
LXX-SS Serum Saver Medium

Buffer
Antibiotics
232 Alsever's Solution 235 Ampicillin (1%)
231 BSA in PBS (7.5%) 372 Ciprobay (2mg/ml)
201 Dulbecco's Phosphate Buffered Saline 228 Fungizone (250ug/ml)
203 Earle's Balanced Salt Solution 208 Gentamicin (1%)
206 Hank's Balanced Salt Solution 212 Kanamycin (1%)
P206 Hank's BSS powder 219 Neomycin (1%)
229 Ringer's Solution 214 Penicillin / Streptomycin (1% ea)
P2XX Salt Mixtures powder 226 Pen / Strep / Fungizone (100x)
234 Transport medium

Additives to Media
250 Amino Acids (MEM) (100xconc) 227 Hepes (1M)
218 Aminopterin (50xconc) 210 HAT (50xconc)
217 Colcemid (10ug/ml) 211 HT (50xconc)
224 DMSO sterile 213 Non-essential amino acids
204 EDTA (0.1%) 207 Sodium bicarbonate (7.5%)
225 Glucose (20%) 215 Sodium Pyruvate (100mM)
209 Glutamine (200mM) 216 Vitamins (MEJM) (100xconc)
230 Glycerol sterile

Growth Factors
Enzymes
A03 Insulin/Transferrin/Selenium ITS 202 Trypsin (0.25)
A01 Linoleate or Flaxoil (2%) on BSA 205 Trypsin/Versene
A04 Transferrin (human)
A06 Insulin (recomb.)

Services Slides
NRBM 25cm flask w X-Cell Line (min.2 flasks) 1204 4 well (4mm) slides (100)
PXX-cm Custom-made powder medium / buffer 1205 12 well (5mm) slides (100)
FILTR Filtration - powder supplied 0806 8 well (6mm) slides (100)
MYCT Mycoplasma testing of cells 0811 8 well (11mm) slides (100)
LXX+ Supplements medium (std. + add) 0406 4 well (6mm) slides (100)
401 Water for biotechnology (2l) (min. 20 liter)

STERILE PLASTIC WARE FOR CELL CULTURE AVAILABLE FROM HIGHVELD BIOLOGICAL

Petri dishes Cell culture flasks
83.1800 35 x 10mm 83.1810 25cm/50ml canted
83.1800.00 35 x 10mm with grid 83.1813 75cm/250ml canted
83.1801 60 x 15mm 83.1812 175cm/650ml canted
83.1801.001 60 x 15mm with grid
83.1802 100 x 20mm
83.1803 150 x 20mm

Cell scrapers
25cm w moving head

Well plates with lids
Serological pipettes (indiv. wrapped)
96-well 0.38ml, flat 86.1251.001 1ml - yellow
24-well 3.6ml, flat 86.1252.001 2ml - green
6 well, flat 86.1253.001 5ml - blue
86.1254.001 10ml - orange
86.1256.001 25ml - red

Microtiter plates w separate lids or foil
Round bottom tubes
82.1581 96-well, flat
82.1582 96-well, round bottom
82.1583 96-well, conical bottom 55.476.013 PS 75x12, 5ml plain
82.1584 Lids for plats 55.526.006 PP 75x12, 5ml plain
82.1586 Adhesive foil 62.515.006 PP 100x16, 13ml grad

Transfer pipettes Conical tubes
86.1180.005 1ml, 17-22ul/drop 62.547.254 PP 114x28mm, 50ml
86.1172.005 3.5ml, 30-45ul/drop 62.553.542 PS 120x17mm, 15ml
86.1175.005 6ml, 20-30ul/drop 62.554.502 PP 120x17mm, 15ml

Microtubes Colored screw caps for microtubes
72.692.005 1.5ml, no print 65.716.001 Blue
72.693.005 2ml, no print 65.716.003 Red
72.694.005 2ml, skirt, writing, grad. 65.716.005 Green
72.730.005 0.5ml, skirt, no print 65.716.006 Orange
72.730.006 0.5ml, skirt, writing, grad. 65.716.008 Violet