HiKeratinoXLTM Keratinocyte Expansion Medium, Serum Free

Product#: HM-AL524
$237.07
HM-AL524
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HiKeratinoXL™ Keratinocyte Expansion Medium, Serum free

Product Code: HM-AL524



Product Description:

HiKeratinoXLTM Keratinocyte Expansion Medium is a serum free medium used for in vitro cultivation and expansion of Human Adult Epidermal Keratinocytes (HAEK) and Human Epidermal Keratinocytes from Juvenile Foreskin. It contains basal medium (Part A), keratinocyte growth supplement (Part B) and Bovine Pituitary Extract (Part C). Part A consists of inorganic, organic salts, amino acids, vitamins and sodium bicarbonate. Part B consists of growth factors and nutrients necessary for growth of keratinocytes. This medium and supplement is devoid of antibiotics and antimycotics.

Products Required But Not Supplied

1.Media Supplements Code
Antibiotic-Antimycotic Solution 100X
[or] Gentamicin
Amphotericin B solution 1000X
A002

A031
2.Reagents for Sub-culture Code
Dulbecco’s Phosphate
Buffered Saline (DPBS)
TL1006
Trypsin/EDTA Solution 1X TCL128
Trypan Blue 0.5% solution TCL005
Soyabean Trypsin Inhibitor TCL068
3. Reagent for Coating Culture Vessel Code
1% Collagen Solution in DPBS TCL127

Directions:

1. Thaw melanocyte growth supplement (Part B) overnight at 2-8 oC. Note: Precipitates in Part B after thawing are normal. Precipitates will not affect the performance of the medium.
2. Disinfect the external surface of the bottles of part A and Part B by spraying with isopropyl alcohol before placing in a bisafety hood.
3. Transfer the entire content of Part B to basal medium 
(Part A) under aseptic condition. Note: If desired, 5ml of antibiotic-antimycotic solution (A002) can be added to 500ml of complete medium.
4. Tightly cap the bottle and swirl gently to ensure proper mixing. Note: Do not mix vigorously. Doing so will cause formation of foam.
5. Store the complete medium at 2 - 8 oC until use

Quality control:
  • Appearance:Part A: Light pink coloured clear solution Part B: Pale yellow coloured hazy solution
  • pH of Part A: 7.00-7.60
  • Osmolality in mOsm/Kg H2O of Part A: 325.00-365.00
  • Sterility:No bacterial or fungal growth is observed after 14 days of incubation, as per USP specification.
  • Cultural Response:The medium is tested for optimal cell growth and proliferation of human mammary epithelial cells.
Storage and shelf life:

Store Part A at 2-8 oC away from bright light. Store Part B at -20 oC. Use before expiry date given on the product label. Shelf life of the complete medium is 4 weeks at 2-8 oC.
Note: Freezing of the basal medium and complete medium is not recommended. Avoid repeated freezing and thawing of the growth supplement

Table 1 : Collagen Coating of Culture Vessel
  Key Points to Remember Time Required (approx.)
  For uniform coating , make sure that the incubator is properly levelled  
Aseptically add 0.5% gelatin solution (TCL109) Refer Table 3 for recommended volumes of gelatin solution 1 min
Incubate overnight 37oC at incubator   2 hrs
Aspirate gelatin solution with the help of pipette    
COLLAGEN COATED CULTURE VESSEL IS READY FOR USE
If the vessel is not to be used immediately, store at 2-8 oC upto one week. Flasks should be kept with caps tightly closed and plates should be sealed with a parafilm during storage  
 
Table 2 : Recommended Volumes of Gelatin Solution for Different Culture Vessels
Culture Vessel Volume Per Well
96-well plate 75 µl
48-well plate 150 µl
24-well plate 300 µl
12-well plate 500 µl
6-well plate 1 ml
T-25 Flask 5 ml
T-75 Flask 10 ml


Table 3 : Protocol for Thawing
• Cryopreserved cells are supplied in liquid nitrogen dry vapor shipper (-150oC to -130oC).
• Upon receipt, immediately transfer the vial to the vapor phase of liquid nitrogen tank.
• Store it in the tank until further use. Cells must be processed at least in a BSL II hood.
    Key Points to Remember Time Required (approx.)
1. Preparation of Culture Vessel
a. Add 5ml of complete medium to a T-25 flask Preparation of complete medium AL533 (Part A 500 ml) + (Part B 25.5 ml) + A002 (5 ml) [For details, refer Technical Data Sheet of AL533] 60 secs
b. Place the flask at 37°C to equilibrate the medium Flask leaking or broken Discard in bio-hazardous waste, if required Intact flask Remove the flask from plastic bag 30 mins
2. Thawing Procedure                                                                     Make sure water bath is set at 37oC before starting the thawing procedure
a. Remove cryovial from the liquid nitrogen tank/ shipper wearing appropriate protective gear Thawing should be AS FAST AS POSSIBLE to minimize cell damage  
b. Immediately thaw the vial partially by holding in a water bath at 37°C DO NOT hold the vial in water bath for more than 90-120 secs
AVOID getting water up to the cap of the vial
90-120 secs
c. Disinfect the vial by swabbing thoroughly with 70% isopropyl alcohol   10 secs
d. Add the cell suspension drop by drop to the T-25 flask containing the pre-warmed complete medium. Keep swirling the flask while adding the cell suspension DO NOT centrifuge cell suspension
Dropwise addition is required to prevent the cells from stress induced by exothermic reaction
30-60 secs
e. Cap the flask and shake gently to ensure proper mixing and uniform distribution of cells in the medium   10 secs
3. Incubation
a. Incubate the cells at 37°C and 5% CO2 Check for cell attachment in 2-3 hrs 2-3 hrs
b. If more than 70-80% cells are attached, replace the medium with fresh medium Medium change after 2-3 hours is mandatory to remove traces of DMSO. If c ells have not attached, centrifuge the cell suspension at 1000 rpm for 7-8 mins, resuspend and seed in fresh medium 60-120 secs




7-8 min
c. Incubate the cells at 37°C and 5% CO2   3-5 days
YOUR CELLS ARE READY TO SUB-CULTURE
4. Maintenance
a. Monitor the cells every day
b. Change the medium every alternate day
c. Sub-culture once cells reach 70 - 80% confluence
  Use the recommended freezing medium for cryopreservation of cells.
DO NOT allow the cells to reach 100% confluency before sub- culture or cryopreservation.
In case of reduced serum or serum free media, use trypsin inhibitor solution (TCL068) for neutralization of Trypsin during subculture. Usage of just medium for neutralization will result in inefficient neutralization and will stress the cells resulting in reduced viability and cell death.
 

Table 4 : Sub-culture
• HAEK/Human Epidermal Keratinocytes from Juvenile Foreskin can be sub-cultured at a seeding density of 5000-10,000 cells/cm2 .
• Sub-culturing ratios can vary from 1:2 - 1:5
• A confluent T-25 flask of HUASMC yields 1.0 x 106 cells
    Key Points to Remember Time Required (approx.)
a. Aspirate entire medium and discard DO NOT disturb the monolayer   60 secs
b. Wash the cells with 2-3 ml DPBS to remove residual medium

c. Aspirate off the DPBS and discard
Prior to use, make sure that TrypsinEDTA solution is equilibrated to room temperature 60 secs
d. Add 2 ml pre-warmed TrypsinEDTA (TCL128) solution

e. Incubate the flask at 37oC for 1 min

f. Check for rounding of the cells and keep tapping the flask gently for 1 min
 
Gently rock the flask to ensure complete coverage of the TrypsinEDTA solution over the cells

Exposing the cells to Trypsin- EDTA for longer time leads to loss of cell viability

This will allow complete dissociation of cells
1 min




1 min
g. To neutralize action of trypsin add 4 ml of TCL068


h. Transfer contents of flask to 15 ml centrifuge tube using pipette

i. Centrifuge the cells at 1000 rpm for 10 min i


Very small pellet will be observed





15 secs


10 minD




 
j. Carefully discard the supernatant by aspiration

k. Add 2 ml of AL524

l. Resuspend the pellet by pipetting gently to get a homogenous mixture
 
DO NOT POUR


Vigorous pipetting will stress the cells
60 secs
m. Count cells using hemocytometer

n. Seed at recommended seeding density in a new flask containing fresh complete medium Refer to Table 5
DO NOT refrigerate cells after splitting Seed immediately
 
10-15 mins
o. Incubate in a humidified incubator at 37ºC and 5% CO2   48 hrs
Maintenance
a. Monitor the cells every day   Upto 50% Confluency: Change the medium on alternate day After 50% Confluency: Change the medium everyday
b. Change the medium every alternate day
c. Sub-culture once cells reach 70 to 80% confluence

Table 3 : Seeding Density
Flask Recommended Seeding Density No. of Cells Per Flask Volume of Medium (ml)
T-25 5000 cells/cm2 0.125 x 106 5 - 7
10,000 cells/cm2 0.25 x 106 5 - 7
These are recommended seeding densities from literature and our studies. Higher seeding densities do not cause any harm to the cells and reduce the required population doublings per passage. Lower seeding densities may cause cells to lose viability, detach during culture and in general take more population doublings to reach confluence
 
 

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