Stem cell technology, explained: from isolation to the freezer

A bag of cord blood in a nitrogen tank and a dose of an approved cell therapy share one awkward fact: both are alive. Every step between donor and freezer can kill cells, contaminate them or change what they do. To follow one product from start to finish, read how a cell therapy is made, from donor to dose.

Where the cells come from

Bone marrow

Marrow holds blood-forming (hematopoietic) stem cells and a smaller group of stromal cells that support them. Marrow is drawn by needle from the hip bone under anesthesia, though many donors now give stem cells from the bloodstream by apheresis instead. NMDP explains both routes, as does our piece on what blood stem cell donors go through. The stromal cells, called mesenchymal stromal cells or MSCs, start a different family of products, covered in our explainer on bone marrow stromal cells.

Cord blood

After a birth, the blood left in the umbilical cord and placenta can be drained into a sterile bag. It is rich in blood-forming stem cells, and cord blood transplants tolerate a less exact tissue match than adult marrow, which is why it has been used since 1988. The catch is volume: one collection is small, which limits the dose for adults. Our cord blood banking explainer covers how units are collected and stored.

Adipose tissue

Fat removed by liposuction contains some MSC-like cells. Breaking the tissue down with enzymes and spinning it yields a mixture called the stromal vascular fraction. Fat is easy to collect in bulk, one reason clinics like it.

Induced pluripotent stem cells

In 2006 Shinya Yamanaka's lab showed that mouse skin cells could be returned to an embryo-like state by switching on four genes, and in 2007 the same was done with human cells. Yamanaka shared the 2012 Nobel Prize for the work. Induced pluripotent stem (iPS) cells can in principle become any cell type. The catch is safety: a cell still pluripotent in the final product could form a tumor. Our iPS cell explainer goes further.

Isolation and sorting

A raw collection is mostly cells nobody wants. Separation happens in stages.

Density gradients

Blood or marrow is layered on a liquid of carefully chosen density and spun in a centrifuge. Red cells and most granulocytes sink through. Mononuclear cells, which include the stem cells, collect in a thin cloudy band at the boundary, where they can be drawn off. Cord blood banks use a related, often automated, step to shrink each unit before freezing.

Markers and flow cytometry

Cells carry surface proteins that work like name tags, labeled with CD (cluster of differentiation) numbers. Blood-forming stem and progenitor cells carry CD34, and the CD34 count is one of the main figures on a cord blood unit's report.

To count cells by marker, a lab adds fluorescent antibodies and runs the cells in single file past lasers in a flow cytometer, which can also sort them. At clinical scale, antibody-coated magnetic beads held in a magnetic column are more common.

MSCs have no single defining marker. A 2006 position statement from the International Society for Cellular Therapy set minimum criteria still in wide use: the cells stick to plastic, carry CD73, CD90 and CD105, lack blood cell markers such as CD45 and CD34, and can become bone, fat and cartilage cells in a dish. Meeting them shows a population is MSC-like, not that it will help a patient.

Growing more cells

Products that need more cells than one donor provides are expanded in culture.

Media, growth factors and scaffolds

Cells grow in a liquid medium of sugars, amino acids, salts and vitamins. Fetal bovine serum was the standard additive for decades; clinical makers increasingly use human platelet lysate or serum-free media, which vary less between batches. Growth factors suit the cell type: fibroblast growth factor for MSCs, and stem cell factor, thrombopoietin and FLT3 ligand for blood-forming cells.

MSCs need a surface to cling to. Small batches grow in stacked plastic flasks. Larger ones go into bioreactors, either stirred tanks where cells grow on tiny beads called microcarriers, or hollow-fiber systems where cells line thin tubes as medium flows past. Tissue engineering adds scaffolds, frameworks of collagen or degradable polymer that give cells a shape to grow into.

Why expansion is hard

Blood-forming stem cells tend to mature as they divide in culture, and mature cells can't rebuild a marrow for years. Raising numbers while keeping that ability is the central problem in cord blood expansion. Omisirge, approved by the FDA in April 2023, adds nicotinamide (a form of vitamin B3) to the culture to slow that drift. Expanding cord blood stem cells covers it and other approaches.

MSCs hit a different wall. After a number of passages (each split into fresh flasks), they slow down and enter a state called senescence. Each extra round also adds handling, contamination risk and a small chance of genetic change.

Testing before release

You can't test a cell product like a pill, because the active ingredient is alive. Release testing asks five questions instead.

• Sterility: cultures for bacteria and fungi, plus tests for mycoplasma and endotoxin. Standard cultures take about two weeks, so a product that lasts only hours may be given before the final result, with a procedure to alert the doctor if it turns positive.
• Identity: the cells are what the label says, shown by marker profile and, for donor products, tissue typing.
• Purity: unwanted cells and leftover reagents stay below set limits. For iPS-derived products, that includes leftover pluripotent cells.
• Viability and dose: the share of living cells, measured with dyes only dead cells take up, and the count of the cells that matter.
• Potency: a measurable sign the product does its job. Blood-forming cells can be grown in a dish to see whether they form colonies. For MSCs potency is harder to define, and it has been a recurring issue in FDA reviews.

Freezing and thawing

Water is the danger. Ice crystals pierce membranes, and as ice forms around cells, the leftover liquid turns salty enough to dry them out. Cryopreservation counters both in three ways.

1. A cryoprotectant. Dimethyl sulfoxide (DMSO), often at about 10 percent of the final mix, enters cells and lowers the temperature at which ice forms. It is mildly toxic at room temperature, so freezing follows soon after.
2. Controlled cooling. A controlled-rate freezer lowers the temperature by about 1 °C per minute, slowly enough for water to leave the cells gradually, and compensates for the burst of heat released as the sample freezes.
3. Deep cold. Bags go into liquid nitrogen tanks, usually held in the vapor above the liquid, at colder than about minus 150 °C. Vapor lowers the risk of contaminants spreading between bags. Researchers have recovered working cord blood stem cells from units stored for more than 20 years.

Thawing has to be fast, usually in a 37 °C water bath or dry thawing device, because slow warming lets ice crystals regrow. The DMSO is then diluted or washed out, since infusing it can cause nausea, flushing and a garlic-like breath odor.

Manufacturing rules and the FDA

What GMP looks like in a cell lab

Good manufacturing practice (GMP) keeps a product consistent and traceable: cleanrooms with filtered air, gowned staff at biosafety cabinets, routine sampling of surfaces and a batch record for every step. For products made from a patient's own cells, the chain of identity (proof that the right cells go back to the right person) matters as much as sterility. Our piece on a day in a cell therapy manufacturing lab shows it hour by hour.

361 and 351 products, in plain words

The FDA regulates human cells and tissues under 21 CFR Part 1271, in two tiers named after sections of the Public Health Service Act.

A section 361 product is regulated mainly to prevent the spread of disease: the establishment registers, screens and tests donors and follows good tissue practice, but needs no approval before marketing. It must meet every one of these conditions:

• It is minimally manipulated, so processing doesn't alter the cells' original relevant characteristics.
• It does the same basic job it did in the donor (homologous use).
• It is not combined with another drug or device, apart from limited exceptions.
• Either it has no systemic effect and doesn't rely on living cells' metabolism, or it goes back to the same person, a close blood relative, or is used for reproduction.

Anything that misses a condition is a section 351 product, a biological drug that needs an investigational new drug application (IND) for testing in people and a biologics license before sale. Expanded cells, iPS-derived cells and CAR-T cells fall here. So do public cord blood units for unrelated patients, which is why licensed products such as Hemacord exist.

This is where many clinics go wrong. FDA guidance finalized in 2017 calls fat processed into stromal vascular fraction more than minimally manipulated, which puts it in the 351 tier. In the FDA's view, a clinic selling such cells without an IND or license is offering an unapproved drug. The agency's consumer alert on regenerative medicine products and its list of approved cellular and gene therapy products are the pages to check.

The steps at a glance

The quickest check on any stem cell product is to ask for its FDA license or IND number. An approved product appears on the FDA's list. A real trial will be on ClinicalTrials.gov, though a listing alone proves little: some clinics register patient-funded studies there too.