Lonza Walkersville, Inc.
Walkersville, MD 21793-0127 USA
U.S. Scientific Support: 800 521 0390
scientific.support@lonza.com
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www.bioscience.lonza.com© 2024 Lonza Walkersville, Inc.

Human CD34+ Cells
Technical information and instructions for use

CD34+ selected cells are a highly purified populationof progenitor cells and allow for greater cell
expansion and differentiation along multiple
pathways. CD34+ and CD133+ progenitors candifferentiate into a number of precursor andspecialized cell types.

Safety StatementsThese products are not for use in GMP
manufacturing, nor human or animal in vivo use,
including use as a diluent or as an excipient, or for
diagnostic use.
These products are for research use only.
WARNING: LONZA PRIMARY CELLS CONTAINHUMAN SOURCE MATERIAL; TREAT AS
POTENTIALLY INFECTIOUS. Each donor is tested
and found non-reactive by an FDA-approved method
for the presence of HIV-I, hepatitis B virus andhepatitis C virus. Where donor testing is notpossible, cell products are tested for the presence ofviral nucleic acid from HIV-1, hepatitis B virus, andhepatitis C virus. Testing cannot offer complete
assurance that HIV-1, hepatitis B virus, and hepatitis
C virus are absent. All human-sourced products
should be handled at the biological safety level 2 tominimize exposure to potentially infectious products,
as recommended in the CDC-NIH manual, Biosafetyin Microbiological and Biomedical Laboratories, 5thedition. If you require further information, pleasecontact your site safety officer or Scientific Support.

Hematopoietic progenitors express high levels of thecell surface glycoprotein CD34. As these cells
mature and differentiate, the levels of CD34
decrease. CD34+ cells can initiate long-term
hematopoiesis and are critical in the drug-discovery
process.
Lonza offers CD34+ progenitors isolated frommononuclear cells using positive immunomagnetic
selection. CD34+ progenitors are available cryopreserved in quantities starting from ≥100,000
cells/order. Purity (as determined by flow cytometry)for bone marrow, cord blood, and mobilizedleukopak-derived CD34+ progenitors is ≥90%.
For answers to frequently asked questions andcitations regarding these products, please visit ourKnowledge Center: https://knowledge.lonza.com

I. Introduction
Lonza offers highly purified CD34+ progenitor cells
isolated from cord blood, bone marrow, andmobilized leukopaks. Cord blood is obtained directlyfrom the umbilical vein either before or after deliveryof the placenta. Bone marrow is obtained fromnormal donors by bilateral aspirates of the posterior
iliac crest. Mobilized leukopaks are collected fromadult blood donors via apheresis. Donors arescreened for general health, normal blood counts,
and infectious diseases as part of the collection
process.

II. Suggested Reagents(Components sold separately)
Medium and Supplements:
1. X-VIVO® 20 Hematopoietic Growth Medium –1 L (Lonza Catalog No. 04–448Q) with ITES
(ThermoFisher GIBCOTM 51500056 or similar)
OR
2. IMDM (Iscove’s Modified Dulbecco’s Medium) –500 mL (Lonza Catalog No. 12–722F) with

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HyClone™ Fetal Bovine Serum U.S. Origin(Cytiva Catalog No. SH30071.01) or similar

2. Recombinant Human SCF (ThermoFisherCatalog No. PHC2111 or similar)
3. Recombinant Human FLT-3 Ligand (SigmaAldrich Catalog No. SRP3044 or similar)

Cytokines for Proliferation:
1. Recombinant Human TPO (ThermoFisherCatalog No. PHC9511 or similar)
III. General Cell Information and Quality Control
Cat. No.

Description

Cells/Vial

2M-101

Cryopreserved Bone Marrow CD34+ Cells≥100,000 viable cells

2M-101A

Cryopreserved Bone Marrow CD34+ Cells≥300,000 viable cells

2M-101B

Cryopreserved Bone Marrow CD34+ Cells≥500,000 viable cells

2M-101C

Cryopreserved Bone Marrow CD34+ Cells≥1 million viable cells

2M-101D

Cryopreserved Bone Marrow CD34+ Cells≥2 million viable cells

2C-101A

Cryopreserved Cord Blood CD34+ Cells≥500,000 viable cells

2C-101B

Cryopreserved Cord Blood CD34+ Cells≥100,000 viable cells

2C-101

Cryopreserved Cord Blood CD34+ Cells≥1 million viable cells

2C-101X

Cryopreserved Cord Blood CD34+ Cells≥1 million viable cells, ≥2 vials

2C-101H≥1 million viable cells

2Y-101C

Cryopreserved Cord Blood CD34+ Cells w/HLA
characterization
Cryopreserved Cord Blood CD34+ Cells w/HLA
characterization
Cryopreserved Mobilized Peripheral Blood CD34+ Cells

2Y-101D

Cryopreserved Mobilized Peripheral Blood CD34+ Cells≥5 million viable cells

2Y-101E

Cryopreserved Mobilized Peripheral Blood CD34+ Cells≥10 million viable cells

2Y-101F

Cryopreserved Mobilized Peripheral Blood CD34+ Cells≥25 million viable cells

2C-101HX≥1 million viable cells, ≥2 vials≥1 million viable cells

Characterization: Total cell population is ≥90% CD34+ as determined by flow cytometry.
Recommended Culture Medium: X-VIVO® 20 Medium (w/ITES) or IMDM (w/15% FBS).
All cells are performance assayed and test negative for HIV-1, Hepatitis-B, and Hepatitis-C. Certificates ofAnalysis (COA) for each product are found on the Lonza Biosciences website. Please see Section IX (Productwarranty and safety, Page 4) for more information on product warranty and performance guarantees.
IV. Unpacking and Storage Instructions

V. Preparation of culture media

1. Check all containers for leakage or breakage.

NOTE: Expansion of cells may lead to decreased
CD34+ expression

2. For cryopreserved cells: Remove cryovials fromthe dry ice packaging and immediately place intoliquid nitrogen storage. Alternatively, thaw anduse the cells immediately. If no dry ice remains,
please contact Customer Service.
3. X-VIVO® 20 Medium instructions: Store medium
at 2°–8°C in the dark. Do not freeze.

1. Decontaminate external surfaces of all vials andthe medium bottle with ethanol or isopropanol.
2. To formulate X-VIVO® 20 Medium (w/ ITES) or
IMDM (w/15% FBS) for culturing CD34+ cells,
add SCF to a final concentration of 25 ng/mL,
TPO to a final concentration of 50 ng/mL, andFLT-3 to a final concentration of 50 ng/mL.

Page | 2 of 53. After cytokines are added to basal medium,
store at 2°–8°C and use within 1 week. Do notfreeze medium.

a. Lonza strongly recommends using ahemocytometer and 0.4% Trypan Bluesolution in a 1:1 ratio; e.g., 20 µL cellsuspension and 20 µL Trypan Blue solution.
Combine both in a separate microcentrifuge
tube and mix with the pipettor. Pipet 10 µLof mixture into each chamber of thehemocytometer and perform a cell count.

NOTE: If there is concern that sterility wascompromised during the supplementation process,the entire newly prepared culture medium may be
re-filtered with a 0.2 μM filter to assure sterility.
Routine re-filtration is not recommended.

i. Adjust the volume of media used toresuspend cells and/or the volume ofTrypan Blue solution added so thatbetween 100 and 500 cells are present in
the hemocytometer grid.

VI. Thawing of Cells
1. Warm 10 mL of X-VIVO® 20 Medium (w/ ITES)
or IMDM (w/ 10% FBS) to room temperature in a
15 mL conical tube.
2. Wipe the outside of the cryovial with 70%
ethanol or isopropanol.
3. In a biosafety hood, twist the cap a quarter turnto relieve internal pressure, and then retighten.
4. Quickly thaw the vial of frozen cells in a 37°C
water bath for no more than 2 minutes until asliver of ice remains. Wipe the outside of the vialwith 70% ethanol.

NOTE: Automated cell counters may be usedinstead of a hemocytometer; however, it is criticalthat the cell counter is calibrated against both live
and dead cell controls prior to use. Lonza strongly
recommends establishing an SOP outlining thecorrect start up, calibration, reagent concentrations,and dilutions for your individual automated cellcounter that has been validated against
hemocytometer counts to ensure accuracy andconsistency. Failure to do so can result in inaccurate
measurements of cell count and/or viability.

NOTE: In the following steps, it is important to work
quickly to ensure high cell viability and recovery. Do
not thaw more than 4 amps at the same time.

NOTE: Lonza strongly recommends verifying cellcounts that fall below advertised specifications on ahemocytometer if those results were determinedusing an automated cell counter.

5. Transfer contents of the vial to the 15 ml conicaltube of medium from step 1, taking care tominimize the pipetting of cells incryopreservation medium as they are fragile.

11. Adjust final volume of cell suspension as
appropriate for downstream applications.

a. Perform this step rapidly! Do not keep cells
in original cryopreservation medium for an
extended period of time. This can reduce
post-thaw recovery and viability.

VII. Initiation of Culture Process andMaintenance
NOTE: This procedure is a recommendation only.
Lonza CD34+ cells are not quality control tested forproliferation, and proliferation is not guaranteed
under the cell warranty. Expansion of cells may leadto decreased CD34+ expression.

6. Rinse the vial with approximately 1 mL ofmedium from the 15 mL conical tube and add itback to the same 15 mL tube.
7. Centrifuge at 300xg for 10 minutes at 2°–8°C.
NOTE: Cell loss of up to 30% can be expected fromthe wash procedure.
8. Carefully aspirate supernatant, removing as
much residual medium as you can without
disturbing the cell pellet.
9. Add 1 mL of cell culture medium (X-VIVO® 20with ITES or IMDM w/15% FBS) to the 15 mL
tube. Gently flick the tube to resuspend thepellet.

1. Seed cells in the complete X-VIVO® 20 Medium(w/ ITES) or IMDM (w/ 15% FBS) at a density of1 x 105 cells/mL.
NOTE: For expansion, the culture medium should
contain additional cytokines listed in Section VI.
2. Replace the media every 3–4 days.
3. Maintain cultures between 0.1–1.5 x 106
cells/mL.
4. Passage cells 2 times per week.

10. Perform a cell count.

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a. Centrifuge cells at 300xg at room
temperature for 10 minutes.

CD34+ Cell Culture Media/Supplements:Cat. No.

b. Carefully remove supernatant without
disturbing cell pellet.

ProductDescription

VIII. Ordering information

X-VIVO® 20 serum-freehematopoietic cell
medium12–722F
IMDM
500 mL Iscove’s
modified Dulbecco’s
medium
A variety of growth factors may be used, including GCSF, GM-SCF and SCF. Multiple growth factors arerequired for optimum growth.

Visit https://bioscience.lonza.com to place anorder or for further information on the following
products.

IX. Product Warrantyc.

04–448QResuspend cells in fresh media and performa cell count (see Section VII, steps 8–11).

d. Reseed cells at density of 1 x 105 cells/mL.

X-VIVO® 20Medium

Cultures have a finite lifespan in vitro.
Normal human CD34+ cells:Cat. No.

ProductDescription

2M-101

Cryopreserved Bone
Marrow CD34+ Cells
Cryopreserved Bone
Marrow CD34+ Cells
Cryopreserved Bone
Marrow CD34+ Cells
Cryopreserved Bone
Marrow CD34+ Cells
Cryopreserved Bone
Marrow CD34+ Cells
Cryopreserved Cord
Blood CD34+ Cells
Cryopreserved Cord
Blood CD34+ Cells
Cryopreserved Cord
Blood CD34+ CellsCryopreserved Cord
Blood CD34+ Cells
Cryopreserved Cord
Blood CD34+ Cells
w/HLA characterization
Cryopreserved Cord
Blood CD34+ Cells
w/HLA characterization
Cryopreserved
Mobilized Peripheral
Blood CD34+ Cells
Cryopreserved
Mobilized Peripheral
Blood CD34+ Cells
Cryopreserved
Mobilized Peripheral
Blood CD34+ Cells
Cryopreserved
Mobilized Peripheral
Blood CD34+ Cells≥ 100,000 viablecells≥ 300,000 viable
cells≥ 500,000 viable
cells≥ 1 million viable
cells≥ 2 million viablecells≥ 500,000 viablecells≥ 100,000 viablecells≥ 1 million viablecells≥ 2 million viable
cells per batch≥ 1 million viablecells

2M-101A
2M-101B
2M-101C
2M-101D
2C-101A
2C-101B
2C-101
2C-101X
2C-101H

2C101HX
2Y-101C

2Y-101D

2Y-101E

2Y-101FLonza guarantees the performance of our primarycells up to two years from purchase only if Lonza’s
recommended media and reagents are used
exclusively and the recommended storage and use
protocols are followed. Any modifications made to
the recommended cell systems including the use ofalternative media, reagents, or protocols will void celland media performance guarantees. If you needassistance in selecting the appropriate media,reagents, or protocol, please contact Lonza
Scientific Support.
When placing an order or contacting Lonza ScientificSupport, please refer to the product numbers anddescriptions listed above. For a complete list of all
primary cell and media products, refer to the Lonza
website or the current Lonza catalog. To obtain acatalog, for additional information, or to speak with
Lonza Scientific Support, you can contact Lonza by
web, e-mail, telephone, or mail (See page 1 fordetails).

≥ 2 million viablecells per batch

ITES (GIBCOTM ITS-X; Thermo Fisher 51500056) is a
product of GIBCOTM.

≥ 1 million viable
cells

HyCloneTM FBS (Cytiva SH30071.01) is a product ofCytiva.

≥ 5 million viable
cells≥ 10 million viable
cells≥ 25 million viable
cells

Recombinant Human TPO (Thermo Fisher PHC9511) is a
product of Thermo Fisher Scientific.
Recombinant Human SCF (Thermo Fisher PHC 2111) is a
product of Thermo Fisher Scientific.
Recombinant Human Flt-3 Ligand (Millipore SigmaSRP3044) is a product of Millipore Sigma.

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All trademarks belong to Lonza, and are registered inUSA, EU or CH or belong to third-party owners and areused only for informational purposes. All third-party
copyrights have been reproduced with permission. The
information contained herein is believed to be correct andcorresponds to the latest state of scientific and technicalknowledge. However, no warranty is made, either
expressed or implied, regarding its accuracy or the results
to be obtained from the use of such information and nowarranty is expressed or implied concerning the use of
these products. The buyer assumes all risks of use and/orhandling. Any user must make his own determination andsatisfy himself that the products supplied by Lonza GroupLtd or its affiliates and the information andrecommendations given by Lonza Group Ltd or its
affiliates are (i) suitable for intended process or purpose,(ii) in compliance with environmental, health and safety
regulations, and (iii) will not infringe any third party’s
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licenses. For more details: www.lonza.com/legal.
© 2024 Lonza.
CD-OP021 05/24

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