Dmitrii Ivolgin. Centre of Regenerative Medicine Organization on the

  St. Petersburg
  North-western region
  Moscow
  Helsinki
13,000,000
13,000,000
10,000,000
Rubles
9,000,000
Economical crisis
12,000,000
11,000,000
11,000,000
6,000,000
5,000,000
400,000
II quarter
III quarter
IV
I
quarter
quarter
II quarter
III quarter
Year period
IV
I
quarter
quarter
II quarter
III quarter
Collection, processing
and storage of
different stem cells types
for regenerative medicine
Cells isolation and expansion
for clinical application in
different areas of medicine
Hi-tech diagnostic techniques,
including genetic testings
Quality management
Organizing the activities of CBB in order to
collect umbilical cord blood units which can be
effectively administer in the field of
regenerative medicine.
Utilization of modern CB processing methods
Double centrifugation
Automated
separation
Semiautomated
separation
CB
Processing
method
NC, х106
cell/ml
Double
Centrifug.
(n = 797)
39,2**
Automated
(n = 1012)
43,3**
SemiAutomated
(n = 78)
42,3**
FDA
requirement
s
(2,5293,1)
(12,5- 95)
TNC,
х106
cells
CD34+
Totally
х106 cell/
CD34+,
ml
х106 cells
Viability
(%)
860,0* 73,04**
0,09**
1,95
98,6**
905,3* 81,9**
0,11
2,1
99,2**
(50,02000,0)
(247,11950,0)
TNC
recovery
(%)
(47,3-98,2)
(65,1-97,6)
891,8* 81,7**
(8,5-83,5) (669,0-17
87,1)
≥ 5.0 x
108
unit
HPC-C
(61,7-94,4)
(0,0020,31)
**(0,0050,375)
0,1
(0,003-0,4
)
(0,05-6,65
)
(0,09-7,5)
1,9
(96,6-100,
0)
(96,6-100,
0)
99,1**
(0,07-6,8)
(96,5-100,
0)
≥ 1.25
x 106
unit
HPC-C
≥ 85%
  TNC (befofe and after processing)
  AB0 blood group and Rh-factor
  CD34+ cells (flow cytofluorometry)
  Viability (flow cytofluorometry)
  Markers of infections (ELISA, PCR)
  Sterility (bacteriological tests)
  CFU-assay
  HLA-typing low and high resolution
  Genetic passport
  Analysis of thelomeres length (flow-FISH)
What:
peripheral blood of mother with circulating fetal DNA
How:
Real-time PCR
When:
From 7-10 week of gestation
Sex determination of fetus
Fetal Rh-factor determination
(negative mothers Rh)
Complex of methods is using:
MLPA
MRC-Holland
Multiplex ligation-dependent
probe amplification
Screening of deletions, interlocations and gene
sequence mutations
PCR
Identification of major gene-mutations, verification of MLPAdetected polymorphisms
Sequence analysis
Identification of gene sequence
 Deletions, duplications, etc. which cannot be recognized by
routine karyotyping
 2-5 Mb length
 Variability from 1 to 10th of genes
  22q11 – Di George’s syndrome
  Williams syndrome
  Angelman syndrome
  1p36 syndrome
  Smith-Magenis syndrome
  17q21 syndrome
  Microdeletion of subthelomeric region 4p
  5p-minus syndrome (cat’s cry syndrome)
  Rubinstein-Taybi syndrome
  Langer-Giedion syndrome
Genotype
CCR5
Absolute
number
Relative
number, %
WT/WT
2835
81,33
WT/delta32
612
17, 56
del32/delta32
39
1,12
Totally
3486
100
224 bp
Number of СD34+:
4
8х10
Number of СD34+:
4
1600х10
Bone marrow
Research projects
Adipose tissue
Traumatology
Umbilical cord
blood
Umbilical cord
Autoimmune diseases
(multitiple sclerosis,
diabetes, Crohn's
disease)
Regenerative therapy
(toxic hepatitis)
Date
Status
Insulin
rate
HA1c,
Mmol/L
25.07.2014
Dry mouth, weakness,
excessing sweating
20
85,9
18
71,3
16
54,6
Gain weight +3 kg,
01.08.2014 appetite and general
state improvement
Good general state,
reinitiation of
05.09.14
exersices, working
capability increasing
Before
24 h. after
MSC infusion
1 month after
MSC infusion
Reference range
Т- cells (CD3+ CD19-) rel.
60,2
70,7
61,75
52 – 76 %
Т- cells (CD3+ CD19-) abs.
667
1636
1301
950 – 1800 cells/μL
Т - helpers (CD3+ CD4+) rel.
16,46
36,6
26
31 – 46 %
Т- helpers (CD3+ CD4+) abs.
206,45
847
544
570 – 1100 cells/μL CTL (CD3+ CD8+) rel.
38,84
28,9
34,4
23 – 40 %
CTL (CD3+ CD8+) abs.
487
668
723
450 – 850 cells/μL
Immunoregulatory index (Тh/CТL)
0,4
0,8
0,8
1,4 – 1,7
T-NK cells (CD3+ CD16+ CD56+) rel.
9,6
2,4
4
0,1 – 8 %
T-NK cells (CD3+ CD16+ CD56+) abs.
220
55
89
5 – 200 cells/μL
NK cells(CD3- CD16+ CD56+) rel.
28,76
17,5
19,5
9 – 19 %
NK cells (CD3-CD16+ CD56+) abs.
460,65
404
431
180 – 420 cells/μL
B cells (CD3- CD19+) rel.
10,5
10,3
11,2
6 – 18 %
B cells(CD3- CD19+) abs.
131,7
238
236
150 – 450 cells/μL
T cells CD3+ HLA DR+ (late activation) rel.
20,5
0–5%
T cells CD3+ HLA DR+ (late activation)
abs.
257
0 – 120 cells/μL
MNC
  Intermittent claudication and constant pain at rest
disappeared in 1.5 months
  Numbness and extremity coldness in the area of the
foot went away in 3 months
  After 5 months, there was a pulsation on the dorslim
of foot arteria.
  Patients getting past any distance pain-free
Sex: ♂ - 14
Age: min – 21 y.
♀ - 18
max – 90 y.
DS:
 Dysarthrosis and long-lasting non-healing long bone
fracture;
 Intra-articular displaced condyle of thibia fracture
 closed ankle fracture;
 closed splintered long bone diaphysis fracture;
 Proximal femoral fracture;
 subdermal Achilles tendon injury.
 soft tissue prolapse
 osteoarthrosis
9 months post
fracture –
consolidation of
fracture…
… avasular
necrosis…
6 months post
adipose tissue
MSC application
64 y.o. ♀
6 week
Before
After operation
1 year
12 week
 Fibroblasts of human derma
 Cell line categorized in
Pokrovsky SCB and SIL of
Cell Technologies NorthWest State Medical
University n/a I.I.
Mechnikov
 3-4 days 5-8th passage
liquid culture with 250 000
cells/ml of gel concentration
RESEARCH INSTITUTE OF EMERGENCY MEDICINE
67 patients
( adult 24 –
78 y.o.)
Application: Burns of boiling water, hot
oil, contact and flame burns, trophic sores,
diabetic foot.
Notification: Use of the drug only after
the suppression of vegetating of pathogenic
microorganisms in the wounds and remove
dead tissue
Applying gel with fibroblasts
 to burns (II, IIIa degree)
 on donor sites
 above the area of the mesh graft
 to sores
Thermal burn IIIа degree
Thermal burn IIIа degree
After 7 days of allofibroblasts
applications
After 9 days of
allofibroblasts applications
Transplanted sieve
graft
(coefficient of
plastics 1/2-1/4),
coated with a layer
of gel with
allofibroblasts
After 4 days.
Edema and discharge is
absence
Partial epithelialization
of autotransplatat
perforation zones
After 7 days
epithelialization of
autotransplatat
perforation zones
1. Hydroxyethyl cellulose polymer (non-toxic,
water soluble, does not cause chromosomal
aberrations. The optimum concentration of
the gel - 1% - does not disturb the
adhesive properties of fibroblasts)
2. Allogeneic human fibroblasts (250 000
cell/ml)
3. Fixation on the lesion place – Lomatuell,
Lohmann&Raucher