An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Summary

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Abstract

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug – clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Introduction

Mesenchymal stem cells (MSCs) play a major role in regenerative medicine and tissue engineering. They can migrate, differentiate into various cell types ¹ and engraft, which renders them the ideal candidates for autologous therapies ^2,3. Lately, clinical trials using MSCs for bone and cartilage repair, graft versus host disease or heart disease were launched ⁴. These MSCs can be harvested from the umbilical cord or adipose tissue but most promising results were obtained from bone marrow derived stem cells ⁵.

The iliac crest allows to collect a considerable amount of bone marrow and therefore serve....

Protocol

NOTE: Human bone marrow aspirates from the iliac crest were collected from consenting donors with the approval of the ethics committee of the canton of Lucerne. Canine bone marrow aspirates from the iliac crest were collected with dog owner’s consent.Human (approx. 20 ml) and canine (approx. 10 ml) bone marrow aspirates were anti-coagulated by addition of 15 ml of 3.8% sodium citrate immediately after withdrawal in the operation theatre. The samples were transferred to the laboratory environment for processing the same day as withdrawn.

1. Preparation of Urokinase (Prior to 1^st Use)

1. Reconstitute urokinase using ....

Results

The facts that 74% of canine bone marrow samples (n=54) contained clots when they arrived in our laboratory (Figure 1A) along with decreased MSC yields from these samples, made us believe that a considerable number of MSCs was trapped within the clots. Indeed, a simple DAPI-stain of sectioned clot material confirms the presence of nucleated cells in high density (Figure 1B). This ultimately leads to low numbers of MSCs available for expansion culture, which triggered us to develop the pr.......

Discussion

Routinely we sample bone marrow while the patient is undergoing surgery (in our case mainly spine surgery), with the advantage that only little additional work has to be carried out by the personnel in the operation theatre. Even though the samples are mixed with sodium citrate immediately after withdrawal, many samples were partially clotted when they arrived in the laboratory for processing. At this stage, resampling to replace clotted specimens would be a separate additional intervention necessitating again local or g.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Basal Medium Components
PenStrep 100X	Gibco	15140122
Human FGF-basic	Peprotech	100-18B
MEM Alpha w/ Nucleoside, w/ stable Glutamine	Amimed	1-23S50-I
FBS Heat Inactivated	Amimed	2-01F36-I
Amphotericin B	Applichem	A1907
Adipogenic Medium Components
DMEM-HAM F12 + GlutaMAX	Amimed	1-26F09-I
Insulin	Sigma	I5500
Rabbit serum	Gibco	16120099
Dexamethasone	Applichem	D4902
3-Isobutyl-1-methylxanthine	Sigma	I5879
Biotin	Sigma	B4639
Rosiglitazone	Sigma	R2408
Pantothenate	Sigma	P5155
Oil Red-O	Sigma	O0625
Osteogenic Medium Components
L-ascorbic acid 2-phosphate	Sigma	A8960
ß-glycerophosphate	Sigma	G9422
Silver nitrate (AgNO3)	Sigma	S6506
Chondrogenic Medium Components
Biopad - sponge shaped medical device	Euroresearch
L-proline	Sigma	P5607
Insulin-Transferrin-Selenium X	Gibco	51500056
Human transforming growth factor-β1	Peprotech	100-21
Alcian Blue 8GX	Sigma	A3157
Nuclear fast red	Sigma	N8002
Generic
Tri-Sodium citrate dihydrate	Applichem	A3901
PBS	Applichem	964.9100
Urokinase	Medac	1976826
0.5% Trypsin-EDTA	Gibco	15400054
Giemsa stain	Applichem	A0885
Formaldehyde	Applichem	A0877
Sulfuric acid (H2SO4)	Applichem	A0655
Dimethyl sulfoxide (DMSO)	Applichem	A1584
Magnesium chloride (MgCl2)	Applichem	A3618
Guanidine hydrochloride	Applichem	A1499
Consumables
50 ml reaction tube	Axygen	SCT-50ML-25-S
10 ml syringe	Braun	4606108V
Sterican needle (22G)	Braun	4657624
1.7 ml Microtubes	Brunschwig	MCT-175-C
100 μm cell strainer	Falcon	6.05935
sterile forceps	Bastos Viegas, SA	489-001
sterile scalpel	Braun	5518059
Primaria cell cuture dish	Falcon	353803
C-Chip Neubauer Improved	Bioswisstech	505050
cell culture flask - Flask T300	TPP	90301
Equipment
Microbiological biosafety cabinet class II	Skan	82011500
water bath	Memmert	1305.0377
Stripettes Serological Pipette 5ml	Corning	4487-200ea
microscope	Olympus	CKX41
humidified incubator Heracells 240	Thermo scientific	51026331
Heraeus Multifuge 1S-R	Thermo scientific	75004331