HiFi™ Human Umbilical Vein Endothelial Cells (HUVEC)PRODUCT CODE: CL002

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 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 4): Immediately transfer the

vial to a liquid nitrogen tank upon receipt using forceps. DO NOT

hold the vial in hand. DO NOT freeze 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

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

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

Product Description and ContentsHiFi™ Human Umbilical Vein Endothelial Cells (HUVEC) are isolated

from human umbilical cords collected post-partum.

HUVEC 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: Human

Tissue: Umbilical vein endothelium

Type of Cells: Endothelial

Growth Characteristics: Adherent

PRODUCT INFORMATION

Products Required But Not Supplied1. Growth Medium

HiEndoXL™ Endothelial CellExpansion Medium

Code

AL517 (Reduced Serum)

2. Media Supplements

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

A002

A031

3. Reagents for Sub-cultureDulbecco’s PhosphateBuffered Saline (DPBS)

TL1006

TCL007

TCL005

4. Reagent for Coating Culturevessel

Trypsin/EDTA Solution 1X

EnVzyme™ Easy TCL137Trypan Blue 0.5% solution Trypsin Inhibitor from Soyabean

TCL068

0.5% Gelatin solution in DPBS TCL109

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 flaskRemove 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-CULTURESub-culturing requires gelatin coating of culture vessel [Refer Table 2]

Table 3 : 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 2 : Gelatin Coating of Culture Vessel

Key Points to Remember

Time

Required

(approx.)

Refer Table 3 for recommended volumes of gelatin solution

Overnight

Flask 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

GELATIN COATED CULTURE VESSEL IS READY FOR USE

If vessel is not used immediately, store at 2-8o upto one week

Aseptically add 0.5% gelatin solution (TCL109)

Incubate overnight at 37oC incubator

Aspirate gelatin solution with the help of pipette.

1 min

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 AL517

(Part A 500 ml) + (Part B 20 ml) + A002 (5 ml)[For

details, refer Technical Data Sheet AL517]

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 beAS 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 up to 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. Keepswirling the flask while addingthe cell 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 and resuspend in fresh medium

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

3-5 days

YOUR CELLS ARE READY TO SUB-CULTURE

4. Maintenance

a. Monitor the cells every day Use the recommended freezing medium for cryopreservation of cells

b. Change the medium

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

secs

7-8 min

60-120

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

Table 5 : Sub-culture• HUVEC 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 HUVEC yields approx. 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 ml DPBSto remove residual medium

c. Aspirate off the DPBS anddiscard

Prior to use, make sure that dissociation solution is equilibrated to room temperature

60 secs

d. Add 0.5 ml pre-warmed Trypsin-EDTA solution or 1mLprewarmed EnVzyme™ Easysolution

Gently rock the flask to ensure complete coverage of the dissociation solution over the cells

e. Incubate the flask at 370C

Trypsin-EDTA solution dissociates HUVEC in approx. 30sec

EnVzyme™ Easy solution dissociates HUVEC in approx. 7-10 min

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

30 sec

f. Microscopically monitor theflask

g. When the cells start roundingup, gently tap the flask toensure complete detachmentof cells

15 secs

h. When using Trypsin-EDTA, neutralize its action byadding equal amount of Soyabean Trypsin Inhibitor Solution (TCL068).

j. Pipette gently to get ahomogenous mixture of cells

Vigorous pipetting will stress the cells

60 secs

EnVzyme™ Easy is gentle on cells and longer exposure does not harm the cells. It does not require neutralization

7-10mins

-

-

i. When using EnVzyme™ Easy, add 1mL complete medium

Table 5 : Sub-culture• HUVEC 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 HUVEC yields approx. 1.0 x 106 cells

Key Points to Remember

Time

Required

(approx.)

m. Incubate in a humidified incubator at 37ºC and 5% CO2

48 hrs

Maintenancea. Monitor the cells every day

b. Change the mediumc. Sub-culture once cells reach

70 - 80% confluence

Table 6 : Seeding Density

Flask Recommended Seeding Density 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

k. Count cells using hemocytometer

l. Seed at recommendedseeding density in a new flask containing fresh complete medium. Refer to Table 6

DO NOT refrigerate cells after splitting

Seed immediately

10-15 mins

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

Percentage viability: NLT 80%

Total no. of population doublings: NLT 15

Sterility Testing Mycoplasma: Not detected

Bacteria, Fungi and Anaerobes: Not detected

Virus TestingHIV: Not detected

Hepatitis B Virus: Not detected

Hepatitis C Virus: Not detected

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

Negative Markers (>95% events) Smooth muscle α- Actin: Negative

Marker Analysis Assay By Flow CytometryThis data has been generated on 8 parameter 3 laser Partec CyFlow® Cube 8 Flow Cytometer and is specific for representative batch of HUVEC.

Positive Markers (>95% events) CD31

By Immunocytochemistry Von Willebrand Factor (vWF): Positive

HUVEC immunostained with vWF-FITC antibody (100X)

Table 7 : Related Products

Product Name Code Packing

HiEndoXL™ Microvascular Endothelial Cell Expansion Medium, Reduced Serum

AL527-500ML 1x500 ml

EZXpand™ Human Umbilical Vein Endothelial Cell Culture KitCCK026-0.5CCK026-T25

0.5 million cells/vial 1 T-25cm2 flask

CryoXLTM DMSO, 1XWith FBS and DMSOWithout Antibiotics

TCL043-50ML 1x50 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