ZenBio Technical Support Information: FAQ

Shipping Questions

How are the cells shipped?
Cells cultured in multiple-well plates are filled with medium and sealed using our patented CellPorterTM package method. Flasks are filled with medium and sealed. Cryopreserved cells are shipped on dry ice. All shipments are sent overnight via Federal Express. Please refer to our manuals for additional information.
How are suspension cells shipped to customers?
Suspension cells are shipped in media with cold preservation buffer on wet ice. For more information, see technical information under fresh hepatocyte products for handling of suspension cells.
How are plated hepatocytes shipped to customers?
Plated cells are shipped in media with cold preservation buffer, then vacuum sealed and surrounded by cold packs. For more information, see technical information under fresh hepatocyte products for handling of plated cells.
How do I handle the cells when I receive them?
We provide detailed instructions on handling the cells. We also provide a small amount of the appropriate media for handling with our cryopreserved cells only. Please refer to our manuals for additional information.
How long do I have to wait before receiving the cells?
We do not ship cells domestically on Friday. In general, preadipocytes (in culture or cryopreserved) can be shipped the second day after the purchase order is confirmed. Adipocytes are shipped within two weeks unless we have differentiated cells in stock. Please inquire as to the availability of the adipocytes when ordering.
Does ZenBio deliver cells on nights & weekends?
Yes, but delivery time is also dependant upon customer availability.
If the customer is not available for weekend deliveries, will ZenBio nurture cells over this time period?
Yes, but this will incur a surcharge to the customer.
Upon ordering, how soon can the customer expect delivery of fresh cells?
Suspensions will be shipped as soon as cells are processed and commercial flights are available. Plated cells without Matrigel overlay will be shipped within 24 hours post seeding. The addition of Matrigel overlay requires an additional 24 hours for delivery.

Adipocyte Questions

Where are the cells from?
The preadipocytes are isolated from human subcutaneous or visceral adipose tissue. The adipocytes are differentiated in vitro using these isolated preadipocytes.
How do you differentiate the cells?
The isolated preadipocytes are plated into the culture ware and treated with our Apipocyte Differentiation Medium (DM-2 or OM-DM) to stimulate adipocyte differentiation.
When do the cells differentiate?
Oil droplets should appear within 4-7 days after differentiation is induced. They look extremely small initially. Lipid accumulation continues throughout the first two weeks. The oil droplets gradually fuse to several big locules.
Can I pass the cells?
Adipocytes cannot be passed since they float after trypsinization. Preadipocytes can be trypsinized and replated several times. Preadipocytes grow slower with each passage and differentiate poorly after passage 4. Cells are shipped at Passage 2-4.
How fast do the cells replicate?
The average doubling time is 48-84 hours. However, keep in mind that the replication rate for human preadipocytes varies slightly from patient to patient.
How long do the cells last?
Adipocytes retain similar morphology and express adipocyte specific genes for at least 4 weeks after induction of differentiation. [NOTE: Cultured adipocytes are usually shipped at 2 weeks old.]
Is the beta adrenergic receptor present on these cells?
The subcutaneous adipocytes express beta-1 adrenoceptor and beta-2 adrenoceptor The mRNA for the Β receptor is not detectable by Northern analysis.
Do the cells express leptin? How do you measure it?
Yes. Mature adipocytes (greater than 2 weeks post differentiation) secrete 1-5 ng leptin from 50,000 cells in 24 hours. The leptin level stays the same for 5-6 weeks. Leptin ELISA kits are available from many vendors (R&D systems, Diagnostic Systems Laboratories, etc.). Leptin mRNA is easily detectable by Northern or PCR analysis.
Do the cells respond to insulin treatment?
Yes. Insulin stimulates lipid accumulation within two days. Insulin stimulates both glucose uptake and phosphorylation of IRS1 within 20 minutes.
Can I differentiate the cells myself?
Yes. You can order preadipocytes and pre-made culture media for adipocyte differentiation. The protocol for differentiating the cells can be found in our manual or at our website ( Cell Manuals ).
Do you have cDNA libraries?
Yes. ZenBio and Stratagene, Inc. collaborated to make cDNA libraries of preadipocytes and adipocytes in uniZAP vector. The libraries can be ordered through Stratagene or ZenBio (cat. no. for adipocyte library is #937249 and preadipocyte libraries are #939234 and #937247).
Do you test for pathogens? How?
Yes, ZenBio utilizes a third-party, FDA-certified testing lab to test all samples for HIV 1&2, HTLV 1&2, Hep B & Hep C.
Can I get a test plate of cells?
No. Sample plates are not currently available. Our cells are guaranteed to be viable and responsive to chemical stimuli. Technical support is available Monday. Friday from 9 AM to 5 PM EST at our toll free number 1.866.ADIPOSE. We will make every effort to assist you with any research problems you may experience
Do the cells express the uncoupling proteins? Which ones?
The UCP1 mRNA is detected by PCR after stimulation with PPAR. agonist. UCP2 mRNA is expressed. We do not know if UCP3 is expressed or not.
Can I order visceral adipose?
Yes, we have cells from visceral depots available. Can I request cells from specific depots? Yes, we often have cells from various depots available. Please inquire as to price and availability.
How do I obtain RNA from the cells? How much RNA can I expect?
Use RNA tri-reagent (Sigma) or a guanidinium isothiocyanate solution (Chomcynzski protocol, reference number 4). You can expect approximately 20g total RNA from a 10 cm dish of confluent preadipocytes and 40-60g of RNA from a 10 cm dish of adipocytes.
What patient information do I get?
Donor's sex, age, and BMI will be provided.
Are the cells from one donor?
Yes. We can also provide lot numbers containing cells mixed from 5 to 8 donors to get average responses. Please inquire about availability of single donor and mixed donor lots (called a superlot) at the time the order is placed.

Adult Stem Cells

Can the adult stem cells be differentiated into any additional cell types?
Other researchers have differentiated adult stem cells into muscle, neuronal, and hepatocyte lineages. See references below.
- Mizuno, et al. Myogenic differentiation by human processed lipoaspirate cells. Plastic and Reconstructive Surgery (2002) 109:199-209.
- Seo, et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys. Res. Comm. (2005) 328: 258-264.

Contract assay service

How much does it cost?
It depends on the complexity and size of the contract. Please call ZenBio to obtain specifics and estimated prices. Minimum contracts start at $2,000.
How soon can I expect the results?
Four work days after the assays are terminated.
Why do I contract ZenBio to do the assay?
With our experience and expertise in human adipocyte biology, it would save you time and money if ZenBio conducts the assay. The assay will be repeated at no extra cost if positive controls do not work due to patient variation. We can not ship tissue directly. Experiments using fresh adipose tissue will have to be conducted in ZenBio laboratories.
What is the throughput of these assays?
We can initiate approximately 1,000 assays per day for differentiation or lipolysis assay. Any screening much larger than 10,000 compounds will take a month to prepare.

Fresh Hepatocytes:

What is the difference between a mature population of hepatocytes and an unfractionated population of liver cells?
A mature population of hepatocytes is isolated via percoll density gradients and consists of ~95% large mature hepatocytes. An unfractionated population of liver cells consists of all existing cell types within the liver.
What type of internal quality control is performed on the cells before they are distributed?
Plated cells must be >70% viable at time of plating and >85% confluent at time of shipping. Internal quality control is maintained by keeping a control plate in-house. Suspension cells must be >80% viable at time of shipping.
What happens to cells that do not meet QC standards?
Cells can be used for in-house research projects, academic partners or pilot studies for proof of concept.
What is the normal seeding density of plated hepatocytes?
All plates are seeded to confluency. However, we can seed at specific densities per customer request.
When do plated hepatocytes receive the Matrigel overlay?
Matrigel overlay is added within 24 hours post seeding time and only upon request.
Is it possible to plate suspension delivered hepatocytes?
Yes.
What type of media is used to culture the hepatocytes?
The market accepted DMEM and Williams E medias are used for plating and maintaining hepatocytes in culture. We are willing to address other customer media needs.
Why do fat vacuoles exist in some lot cultures?
The livers received for processing are non-transplantable organs and therefore some can contain fatty deposits.
Are antibiotics used in culture?
Antibiotics are used in culture media, such as penicillin/streptomycin at standard culturing concentrations.
How are tissues screened against diseases?
The organ procurement organizations provide donor serology results. Any conflicting results will be reported to customers within 24 hours.
What is the age range of donor livers received?
We receives donor livers from birth to age 70.

Cryopreserved Hepatocytes:

What is the difference between plateable and non-plateable cryopreserved hepatocytes?
Plateable cryopreserved hepatocytes (Premium Tier 1) must have an attachment efficiency > 70%. Any lot that falls into a lower tier with varying viability and plateability characteristics.
Is it possible to achieve attachment with the non-plateable hepatocytes?
Yes, however the attachment efficiency will range from 0 - 70%.
Is there a way to improve the post thaw viability of the cryopreserved hepatocytes?
Yes, by performing a percoll density gradient, the cell viability can be improved. Refer to the technical information under cryopreserved products.
Why does attachment efficiency vary from lot to lot?
Variability in attachment efficiency is due to variability in donor livers, and therefore has an affect on plateability.

General Blood Products

Blood Product FAQs:

Healthy adults who are at least 17 years old, and at least 110 pounds may donate about a pint of bloodâ€”the most common form of donationâ€”every 56 days, or every two months, depending on iron levels.
Anticoagulants: K2EDTA, K3EDTA, ACD, Sodium Heparin, Lithium Heparin, Sodium Citrate, CPDA-1
Four main red blood cell types: A, B, AB and O. Each can be positive or negative for the Rh factor. AB is the universal recipient; O negative is the universal donor of red blood cells.
Plasma is a pale yellow mixture of water, proteins and salts.
Plasma, which is 90 percent water, makes up 55 percent of blood volume.
42 days: how long most donated red blood cells can be stored when collected in CPDA-1 and stored at 4C. 21 Days is the standard for all other anticoagulants.
Five days: how long most donated platelets can be stored.
All samples are collected in FDA licensed facility and pathogen tested according to FDA guidelines for the following: HBsAG, Anti-HCV, Anti-HIV-1/1, HIV-1 RNA, HBV DNA, HCV RNA, Syphilis

Definitions

Human Serum:
The clear liquid portion removed from a unit of whole blood containing no anticoagulant or additives. Sometimes called: OTC (off the clot) Serum. Average size per unit is 225 mls.
Recovered Human Plasma:
The clear liquid portion removed from a unit of whole blood containing an anticoagulant such as: ACD, CPD, CPDA-1, EDTA, HEPARIN, SODIUM CITRATE (or others). Average size per unit is 225 to 250 mls. . It is generally stored at room temperature or under refrigerated temps. It could be frozen.
Fresh Frozen Plasma:
Plasma from a unit of whole blood frozen within 5 days (120 hours of collection). Also, commonly frozen within 1-8 hours, 1-24 hrs. 1-36 hours, 1-72 hours or 1-120 hrs. (5 days). Good for 1 year for transfusion purposes or fractionation. No expiration for research purposes. Average size is 225 to 250 mls.
Source Plasma:
This is the FDA licensed product name for plasma collected via plasmapheresis. By definition, it must be frozen at -20C or colder and placed at that temperature within 30 minutes. Average size is ~ 880 mls. Most common anticoagulant is 4% Sodium Citrate.
Platelet Rich Plasma:
Plasma removed from a unit of whole blood that has been spun at speed slow enough to keep the platelets still floating around in it. Average size per unit is 200 mls. Shipped at ambient temperature
Platelet Concentrates:
Prepared from a unit of platelet rich plasma, that has been spun at a faster speed to concentrate the platelets toward the bottom of the bag. The excess platelet poor plasma is then removed from the upper portion of the unit, leaving concentrated platelets, in a small portion of plasma. Average size per unit is 50 to 60 mls. Platelet count ranges from 75–200 x 10E9. Handled and shipped at ambient temperature.
Buffy Coat/Leukocytes:
Concentrated white cells obtained from a unit of whole blood. Units can be prepared as bloody: (going deeper into the red cell layer to obtain maximum white cell counts) or clear to pinkish: (not going into the red cell layerâ€¦lowers white cell count, but also reduces red cell population). Average size is 50 to 60 ml. Units can be collected in the anticoagulant of your choice. Average cell count depends on the donor, but ranges from 600-800 million cells.
Whole Blood:
An entire donation of blood collected into the anticoagulant of choice. We collect 450 mls + or - 10% from our donors. When mixed with the anticoagulant in the blood bag. An average unit is 500 to 540 mls.
Packed Cells:
The remaining red portion from a unit of whole blood that has had the plasma removed from it. Average size is 250 to 300 mls.