Colony-Forming Unit Assay Adapted For Patient-Derived Bone Marrow and Peripheral Blood Mononuclear Cells

Summary

This protocol outlines a procedure for inducing differentiation of human hematopoietic stem and progenitor cells derived from patient bone marrow and peripheral blood mononuclear cells. The assay uses a semisolid culture medium supporting erythroid and myeloid lineage commitment and incorporates standardized metrics for reproducible colony classification for preclinical drug characterization.

Abstract

The colony-forming unit (CFU) assay is a foundational technique for studying hematologic diseases, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This assay employs semisolid media supplemented with defined cytokine combinations to support three-dimensional growth and lineage-specific differentiation of erythroid and myeloid progenitor cells. However, patient-derived peripheral blood and bone marrow samples present technical challenges due to reduced viability, increased sensitivity to handling, and variability in colony growth. Furthermore, existing guidelines for identifying and counting colonies show substantial variability in defining colony size and lineage types, limiting reproducibility across studies. The protocol described herein is optimized for the growth and differentiation of hematopoietic stem and progenitor cells isolated from MDS and AML patient samples. The protocol emphasizes gentle cell handling, sterile processing of patient specimens, optimized culture conditions, and growth durations tailored to patient-derived cells. This protocol provides a practical framework for CFU assays using patient-derived samples and establishes a standard metric for consistent colony counting and analysis in hematologic malignancy research.

Introduction

Hematopoiesis is a tightly regulated process in which hematopoietic stem cells (HSCs) residing in bone marrow niches give rise to all mature blood cell lineages. Through a hierarchical series of differentiation events, HSCs generate multipotent progenitors that progressively commit to erythroid, myeloid, and lymphoid lineages before undergoing terminal maturation into functional circulating blood cells. This process is governed by coordinated signaling within the bone marrow microenvironment and by intrinsic transcriptional and epigenetic regulatory mechanisms. Disruptions to these regulatory pathways, particularly through the accumulation of genetic or somatic mutati....

Protocol

This study utilizes de-identified human biospecimens obtained from a biobank under an Institutional Review Board–approved protocol, “Molecular Biology of Benign and Malignant Hematological Disorders” (IRB Protocol #200536), at Albert Einstein College of Medicine/Montefiore Medical Center. All procedures were conducted in accordance with institutional ethics guidelines. All reagents and equipment are listed in the ​Table of Materials.

1. Preparation of patient-derived bone marrow or peripheral blood mononuclear cells

NOTE: According to common patien....

Results

Figure 1 illustrates the colony assay pipeline from patient sample isolation through analysis of colony counts and colony morphology. Figure 2 displays key settings for imaging colonies on a brightfield microscope. Figure 3 and Figure 4 show representative images of colony formation from a single patient-derived sample at 13 days post-plating. In Figure 3

Discussion

The protocol described here outlines an optimized workflow for performing CFU assays using primary bone marrow and peripheral blood samples from patients, with particular attention to maintaining cell viability and ensuring reproducible colony growth. Several steps in the protocol are critical for achieving reliable colony formation when working with patient-derived samples. Rapid thawing of cryopreserved mononuclear cells followed by immediate cooling is essential for preserving cell viability, as prolonged thawing can .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blunt-End 16 G needles	STEMCELL Technologies	28110	for dispensing and plating MethoCult media
Cellometer Auto 2000	Nexelcom Bioscience	26407	cell counter
CHT4 Counting Chambers (cell counter slides)	Revvity	CHT4-SD100-002	slides for cell counter
Dimethyl sulfoxide	Fisher Bioreagents	BP231-100	used for drug reconstitution and cell freezing media
Dulbecco's Phosphate Buffered Saline	Sigma-Aldrich	D8537	for cell culture
Ethanol 200 proof	Decon Laboratories, Inc.	2701	for sterilization of cell culture surfaces
EVOS M7000 imaging system	ThermoFisher Scientific	AMF7000	for colony and plate imaging
Fetal bovine serum	BenchMark	100-106	media supplement for cell culture and freezing
IMDM Modified Culture media	Cytiva	SH30228.01	enriched culture media suitable for patient sample handling
LuerLok Tip 3 mL Syringes	BD	309657	for dispensing and plating MethoCult media
MethoCult H4435 Enriched	STEMCELL Technologies	04435	methylcellulose-based semisolid media for colony assay
Multiwell 6-well plate	FALCON	353046	for colony assay plating
Nikon Eclipse TS2FL Inverted Trinocular Phase Contrast Fluorescence Microscope	Coastal Microscopes	51623	For colony morphology imaging and characterization
Pen Strep	Gibco	15140-122	antibiotic supplement for cell culture
pyrimethamine	Sigma-Aldrich	46706	FDA-approved antifolate
UltraPure Distilled water	Invitrogen	10977-015	for colony assay plating
venetoclax	Selleck Chemical LLC	50-136-6419	FDA-approved chemotherapy
ViaStain AOPI Staining Solution	Revvity	CS2-0106	viability stain for live cell counting