Product Description and Contents

HiFi™ Human Mammary Epithelial Cells are Passage 2 cells

supplied frozen with density of not less than 0.5 x 106 cells

per vial or as proliferating cells in a T-25 culture flask. Each lot

of cells is from a single donor and undergoes growth kinetic

studies and cell marker analysis. Cells are maintained in

antibiotic- free conditions prior to supply.

Source : HumanTissue : Mammary TissueType of Cells : EpithelialGrowth Characteristics : Adherent

PRODUCT INFORMATION

Products Required But Not Supplied1. Growth Medium Code

1. HiEpiXL™ Mammary EpithelialCell Expansion Medium

AL529

2. Media Supplements

Antibiotic-Antimycotic Solution 100X [or Gentamicin-Amphotericin B solution 1000X

A002

A031

3. Reagents for Sub-culture

Dulbecco’s Phosphate Buffered Saline (DPBS)

TL1006

Trypsin/EDTA Solution 1X TCL128

TCL005Trypan Blue 0.5% Solution

Soyabean Trypsin Inhibitor TCL068

4. Reagent for Coating CultureVessel

1% Collagen Solution in DPBS TCL127

HiFi™ Human Mammary Epithelial Cell (HMEC)PRODUCT CODE: CL014

Product WarrantyHiFi™ cells are performance tested using HiMedia media and

reagents. Users must ensure proper handling, storage and use of

the product. We DO NOT GUARANTEE the performance of the cells if protocols and products other than those mentioned in this sheet are used. We are not liable for any damage to the culture arising from improper storage or mishandling.

Initial Handling upon ReceiptFor any damage to the product upon receipt, report it immediately

to the HiMedia marketing representative or technical team.

For any difficulty while handling or processing the cells please

contact HiMedia technical team immediately for flask and within a

month for cryovial after delivery:

022-61169797 / 8767552556 or atc@himedialabs.com

Check List• Proliferating Cells (Refer Table 1) : Cells are supplied in flasks

with closed (non-vented) caps. Loosen one thread of the cap only

after medium change. Use sterile disposable tips and serological

pipettes. Avoid using autoclaved tips and serological pipettes.

• Cryopreserved Cells (Refer Table 2) : Immediately transfer the vial to a liquid nitrogen tank upon receipt using forceps. DO NOT

hold the vial in hand. DO NOTstore the cells at -80⁰C.

COMMON MISTAKESStem cells and primary cells are more sensitive and delicate as compared to the robust continuous cell lines hence, require extreme precautions while handling, subculturing and thawing.

1. Exposing the cryopreserved cells during thawing to warm

temperatures (37oC) for more than 90 - 120 secs

2. Not changing medium within 2-3 hours after thawing

3. Sub-culturing after the cells reach 100% confluence

4. Exposing the cells to trypsin for more than the recommended

time during sub-culturing

Following handling methods can lead to loss of cell viability, alteration in morphology and loss of attachment of cells.

Table 1 : Storage and Handling of Proliferating Cells• Proliferating cells are supplied in a T-25 closed (non-vented cap) flask at room temperature.• The flasks are filled to capacity with transport medium and the neck is sealed with parafilm.• Each flask is individually packed in a plastic bag to contain leakage, if any.

Key Points to Remember

Time

Required

(approx.)

Initial Handling upon Receipt

• Work in areas designated foruse Human origin materialBiosafety Level II (BSL II)

• Wear protective clothing andgloves

→ CHECK• Leakage during transport

Flask leaking or brokenDiscard in bio-hazardous waste if required

Intact flask Remove the flask from plastic bag

→ CHECK• Cell morphology and

confluence• Microscopic visibility of the

cells is hampered due to flaskfilled to capacity

DO NOT discard the medium!120 secs

→ CHECK• Incubate the cells for 3-4 hrs to

allow floating cells to reattachto the surface

DO NOT loosen one thread of the cap! 3 hrs

• Only after incubation, removethe medium and discard

• Add 5-7 ml fresh medium in theflask

• At this stage cells will be clearlyvisible under the microscope

180 secs

• Incubate at 37oC and 5% CO2 Loosen one thread of the cap!1-3 days

YOUR CELLS ARE READY TO SUB-CULTURE

Table 2 : Collagen Coating of Culture Vessel

Key Points to Remember

Time

Required

(approx.)

Aseptically add 1% collagen solution (TCL127)

Refer Table 3 for recommended volumes of collagen solution

1 min

Incubate for 2 hrs at 37⁰C incubator 2 hrs

Aspirate collagen solution with the help of pipette

If the vessel is not to be used immediately, store at 2-8⁰C upto one week.

Flasks should be kept with caps tightly closed and plates should be sealed with a parafilm during storage

For uniform coating , make sure that the incubator is properly levelled

Table 3 : Recommended Volumes of Collagen 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

COLLAGEN COATED CULTURE VESSEL IS READY FOR USE

Table 4 : Storage and Handling of Cryopreserved Cells• Cryopreserved cells are supplied in liquid nitrogen dry vapour 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 mediumto a T-25 flask

Preparation of complete medium AL529 (Part A-500 ml) + (Part B-1.2 ml) + (Part C-2 ml) + A002(5ml)[For detail, refer Technical Data Sheet AL529]

60 secs

b. Place the flask at 37°C toequilibrate the medium

2. Thawing Procedure

a. Remove cryovial from the liquidnitrogen tank/ shipper wearingappropriate protective gear

Thawing should be AS FAST AS POSSIBLE to minimize cell damage

b.DO NOT hold the vial in water bath for more than 90-120 secs

AVOID getting water upto the cap of the vial

90-120secs

c. Disinfect the vial by swabbingthoroughly with 70% isopropylalcohol

10 secs

d. Add the cell suspension dropby drop to the T-25 flaskcontaining the pre-warmedcomplete medium. Keep theflask swirling while adding thecell suspension

Dropwise addition is required to prevent the cells from stress induced by exothermic reaction

30-60 secs

30 mins

Make sure water bath is set at 37⁰C before starting the thawing procedure

Immediately thaw the vialpartially by holding in a waterbath at 37°C

Table 4 : Storage and Handling of Cryopreserved Cells• Cryopreserved cells are supplied in liquid nitrogen dry vapour 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.)

e. Cap the flask and shake gentlyto ensure proper mixing anduniform distribution of cells inthe medium

10 secs

3. Incubation

a. Incubate the cells at 37°C and5% CO2

Check for cell attachment in 2-3 hrs 2-3 hrs

b. If more than 70-80% cells areattached, replace the mediumwith fresh medium

Medium change after 2-3 hours is mandatory to remove traces of DMSO If cells have not attached, centrifuge the cell suspension at 1000 rpm for 7-8 mins, resuspend and seed in fresh medium

c. Incubate the cells at 37°C and5% CO2

3-5 days

YOUR CELLS ARE READY TO SUB-CULTURE

4. Maintenancea. Monitor the cells every day Use the recommended freezing

medium for cryopreservation of cellsb. Change the medium

c. Sub-culture once cells reach 70- 80% confluence

secs

7-8 mins

60-120

Upto 50% Confluency: Change the medium on alternate day After 50% Confluency:Change the medium everyday

Table 5 : Sub-culture• HMEC 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 HMEC yields 1.0 x 106 cells

Key Points to Remember

Time

Required

(approx.)

a. Aspirate entire medium anddiscardDO NOT disturb the monolayer

60 secs

b. Wash the cells with 2-3 mlDPBS to remove residualmedium

c. Aspirate off the DPBS anddiscard

Prior to use,make sure that Trypsin-EDTA solution is equilibrated to room temperature.

60 secs

d. Add 2 ml pre-warmed Trypsin-EDTA (TCL128) solution

Gently rock the flask to ensure complete coverage of the Trypsin-EDTA solution over the cells

e. Incubate the flask at 37°C for 1min

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

1 min

f. Check for rounding of the cellsand keep tapping the flaskgently for 1 min

1 min

g. To neutralize action of trypsinadd 4 ml of TCL068

l. Pipette gently to get ahomogenous mixture

Vigorous pipetting will stress the cells

60 secs

m. Count cells usinghemocytometer

n. Seed at recommendedseeding density in a new flaskcontaining fresh completemediumRefer to Table 6

DO NOT refrigerate cells after splitting

Seed immediately

10-15 mins

This will allow complete dissociation of cells

k. Add 2 ml of AL529

i. Centrifuge the cells at 1000rpm for 10 min

10 min

15 secs

Very small pellet will be observed

j. Carefully discard thesupernatant by aspiration

DO NOT POUR

h. Transfer the entire contents offlask into 15ml centrifuge tube.

Table 5 : Sub-culture• HMEC 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 HMEC yields 1.0 x 106 cells

Key Points to Remember

Time

Required

(approx.)

o. Incubate in a humidifiedincubator at 37ºC and 5% CO2

48 hrs

Maintenancea. Monitor the cells every day

b. Change the medium

c. Sub-culture once cells reach70 - 80% confluence

Table 6 : Seeding Density

Flask Recommended Seeding Densitiy No. of Cells Per Flask Volume of Medium (ml)

T-255000 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.

Upto 50% Confluency: Change the medium on alternate day After 50% Confluency:Change the medium everyday

Growth Performance Assay No. of viable cells/vial: NLT 500,000 cells/vial

Percentage viability : NLT 80%

Total no. of population doublings : NLT 15

WarningThis product is intended for research use only. Not for animal,

human therapeutic or diagnostic use.

Product contains human origin material and should be treated as

potentially infectious. Even if the cells provided have been screened

for viral and bacterial pathogens, human cells may harbor other

known or unknown agents which might pose a health hazard.

Universal handling precautions applicable to biological samples

must be applied as recommended in the CDC-NIH manual.

Quality Control

Marker Analysis Assay Immunoflurescence detection of

Cytokeratin 14 : Positive

Cytokeratin 18 :Positive

Cytokeratin 19 : Negative

Table 7 : Related Products

Product Name Code Packing

HiEpiXL™ Mammary Epithelial Cell Expansion Medium AL529-500ML

CryoXL™ Chemically Defined Serum Free Freezing MediumWith DMSOWithout Antibiotics, Antimycotics and Phenol red

TCL098-50ML 1x50 ml

Gentamicin-Amphotericin B Solution 1000X A031-20MLA031-20ML

1x500 ml

1x20 ml5x20 ml

Disclaimer :

User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and other related

HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the best of our knowledge true and

accurate. HiMedia™ Laboratories Pvt. Ltd. reserves the right to make changes to specifications and information related to the products at any time. Products are not

intended for human or animal diagnostic or therapeutic use but for laboratory, research or further manufacturing use only, unless otherwise specified. Statements

contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd.

A-516, Swastik Disha Business Park, Via Vadhani Indl. Est. LBS Marg, Mumbai - 400 086, India.

Customer Care No. : 022-61471919 | Email : atc@himedialabs.com

Revision: 2/ 2018

Sterility TestingMycoplasma (PCR): Not detectedBacteria, Fungi and Anaerobes as per USP: Not detected

Virus Testing HIV (PCR): Not detected

Hepatitis B Virus (PCR): Not detected

Hepatitis C Virus (PCR): Not detected